dna replication essay conclusion

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DNA Replication

Reviewed by: BD Editors

DNA replication is a process that occurs during cellular division where two identical molecules of DNA are created from a single molecule of DNA. As a semiconservative process, a single molecule containing two strands of DNA in double helix formation is separated, where each strand serves as a template for the new DNA molecules. Because the double helix is anti-parallel and DNA polymerase only synthesizes new DNA from 5′-3′, the template strand reading 3′-5′ results in a continuous, leading strand, while the template strand reading 5′-3′ results in a discontinuous, lagging strand. Being a highly regulated process, multiple proteins are required both during and following replication to quickly correct mistakes and damages.

Deoxyribonucleic acid, known simply as DNA, is the blueprint of all living things. DNA contains genes that code for the physical and metabolic information expressed in an individual while having the potential to be passed down to future offspring. Almost all cells have DNA, which is typically stored in the nucleus. Notable cells that lack DNA include anucleate cells (or cells that lack a nucleus, such as red blood cells). Additionally, some cells may still have DNA despite not having a nucleus, such as with bacterial cells.

DNA Nucleotides

DNA is made up of four building block monomers that are known as nucleotides . Nucleotides consists of three groups:

• A deoxyribose sugar group
• A phosphate group
• A nitrogenous base

All of the nucleotides have the same sugar group and phosphate group, but different nitrogenous bases. The nitrogenous bases in DNA are adenine, guanine, cytosine, and thymine. Adenine and guanine are classified as purines , while cytosine and thymine are classified as pyrimidines . Purines have two rings in their base structures, while pyrimidines have a single ring in their base structures.

(Helpful hint: A simple pneumonic to remember adenine and guanine as purines is “ Pur e A s G old”!)

DNA Structure

Nucleotides are arranged into chains that become individual strands of DNA, which is half of a full DNA molecule. Each strand has a sugar-phosphate backbone that is created when the phosphate of one nucleotide binds to the sugar of the next using a covalent  phosphodiester bond . Specifically, the phosphate is found on the 5′ carbon of one nucleotide, while a hydroxyl group (-OH) is found on the 3′ carbon of the next nucleotide’s sugar group. The -OH of the 3′ carbon is removed, where the phosphate group on the 5′ carbon now also bonds to the 3′ carbon.

The nitrogenous bases stick out from this backbone. A second DNA strand is matched to this first strand based on complimentary base pairing , where a single purine pairs with a single pyrimidine. Specifically, adenine pairs with thymine and guanine pairs with cytosine.  Hydrogen bonds connect the complimentary base pairs, where an adenine- thymine pair has two hydrogen bonds and a guanine-cytosine pair has three hydrogen bonds. A single DNA molecule results in  double helix formation when two DNA strands are matched and bonded.

DNA has directionality that can run either 3′-5′ or 5′-3′ based off of the carbons in the sugar group. The two strands of DNA in the double helix must run opposite to each other in an anti-parallel fashion. Therefore, if the first strand starts at the 3′ end and finishes at the 5′ end, then the second strand must run opposite, starting at the 5′ end and finishing at the 3′ end.

3′- AATCGTAA -5′

5′- TTAGCATT -3′

DNA Replication Is Semiconservative

DNA must be fully replicated before cells divide via mitosis to ensure all daughter cells have identical DNA. It was discovered that DNA replication is semiconservative . In semiconservative replication, the double helix splits into two separate strands. During the replication process, an entirely new strand of DNA is created by using the original template strand and matching the complimentary bases.

DNA Replication Process

Proteins in DNA Replication

DNA replication is highly regulated and requires multiple proteins to run efficiently. A majority of these proteins act as stabilizers and enzymes, with enzymes being proteins that behave as catalysts to create and speed up biochemical reactions.

Some of the major proteins in DNA replication include the following:

Helicase : An enzyme that opens the double helix by breaking the hydrogen bonds between complimentary base pairs

Single-strand DNA- binding proteins (SSBPs) : These proteins stabilize the individual strands of DNA to prevent them from reconnecting.

Topoisomerase : Because unwinding of the DNA by helicase creates tension further down the strand, this enzyme relieves tension by making cuts in the DNA and rejoining them before the replication fork arrives.

Primase : An enzyme that adds a primer (which is a short segment of ribonucleic acid, known as RNA) where DNA polymerase III will attach

DNA polymerase III : An enzyme that creates the new DNA strand by adding nucleotides that are complimentary to the template strand

DNA polymerase I : An enzyme that replaces the RNA primer with DNA

DNA ligase : An enzyme that connects the Okazaki fragments on the lagging strand by closing the sugar-phosphate backbone, creating a single DNA strand

Sliding clamp : A protein that holds DNA polymerase III in place

The Replication Bubble

When DNA begins to replicate, a replication bubble is formed that can be detected visually by electron microscopy. A specific sequence of bases- known as the origin of replication – determines where this replication bubble begins. Inside of the bubble, two Y-shaped replication forks result where DNA is actively replicated on either side of the region. The replication forks are formed as the double strands of DNA are separated by helicase in both directions away from the origin of replication. It is at the replication fork that DNA replication proteins attach to fulfill their functions.

Replicating the Leading Strand

As mentioned previously, DNA strands have an anti-parallel nature, where one strand will run 3’-5’ and the other will run opposite from 5’- 3’. DNA polymerase can only synthesize new strands of DNA in the 5’-3’ direction. In order for DNA polymerase to do this, it must read the template strand from 3′-5′. Therefore, replicating the template strand that runs 3’-5’ results in the synthesis of the  leading strand . The leading strand is a new strand of DNA that is synthesized in a single, continuous chain that starts at the 5’ end and finishes at the 3’ end.

DNA replication of the leading strand when the 3’-5’ template strand is used is as follows:

• The DNA double helix is opened by helicase into individual strands. Topoisomerase relieves the tension further down the double helix.
• SSBPs stabilize the single DNA strands to prevent them from reconnecting.
• An RNA primer is added to the leading strand at complimentary bases by primase.
• DNA polymerase III attaches to the primer. The sliding clamp stabilizes DNA polymerase III.
• DNA polymerase III moves down the leading strand towards the replication fork , adding bases to the new strand from the 5’ end to the 3’ end.

Replicating the Lagging Strand

DNA polymerase can only create new DNA strands from 5’-3’. Therefore, when the 5′-3′ template strand is being replicated- where the new strand must run opposite in the 3′-5′ direction- the new strand cannot be synthesized in a continuous fashion as the leading strand was. To overcome this challenge, additional steps are needed to replicate the 5′-3′ template strand, where this newly synthesized strand is known as the  lagging strand .

In order for the lagging strand to be synthesized, DNA needs to be broken down into smaller segments known as Okazaki fragments . Because of these multiple segments, the lagging strand is also known as the discontinuous strand . By creating these multiple segments, DNA polymerase III is able to synthesize a small portion of the new DNA strand  away from the replication fork in the correct 5′-3′ direction. As the replication fork continues down the double helix in the 3′ direction of the template strand, another Okazaki fragment can be created closer to the fork. DNA polymerase III binds again to synthesize another portion of the new DNA strand away from the fork until it reaches the previous portion already synthesized. These fragments are then connected, resulting in a single DNA strand. This process continues down the entire length of the DNA.

DNA replication of the lagging strand when the 5’-3’ template strand is used is as follows:

• SSBPs stabilize the single DNA strands.
• Primase adds an RNA primer to the lagging strand.
• DNA polymerase III binds to the primer and creates a short segment of newly synthesized DNA from 5′-3′, synthesizing in the opposite direction of the replication fork . This is the first Okazaki fragment.
• As helicase further unwinds the double helix and the replication fork moves down the strand, another primer is added closer to the fork. DNA polymerase III attaches to this primer to synthesize a second Okazaki fragment in the 5′-3′ direction away from the replication fork.
• Once DNA polymerase III reaches the first Okazaki fragment primer, DNA polymerase I removes the primer and replaces them with the proper complementary bases.
• DNA ligase connects the segments of DNA by closing the sugar-phosphate backbone. The two segments are now connected into a single strand.
• This process repeats as the replication fork continues down the length of the DNA.

Prokaryotes vs. Eukaryotes

DNA replication overall is fairly conserved across life. However, general differences exist in the enzymes and mechanisms used , as well as time required between species. The largest differences are between the domains of prokaryotes (bacteria and archaea) and eukaryotes (all other plant and animal cells).

Minor differences between these groups include faster replication time in prokaryotes and shorter Okazaki fragments in eukaryotes. Additionally, prokaryotes only have a single origin of replication, while eukaryotes have multiple origins of replication. The most noteworthy difference between these groups however, is that prokaryotes have circular DNA while eukaryotes have linear DNA. Linear eukaryotic DNA creates an additional challenge that must be regulated. This brings us to telomeres.

Because eukaryotic DNA is linear, they have ends that create a challenge. For the leading strand, DNA polymerase III can continue down the entire length of DNA. However, in the lagging strand, a primer must be added in front of the Okazaki fragment being synthesized before DNA polymerase III can attach and synthesize the new DNA strand opposite of the replication fork. Once the last Okazaki fragment is synthesized, a small DNA segment is leftover at the tip of the strand. This segment cannot be left unattended . If this DNA isn’t replicated, then genetic material will be lost each time replication occurs. After several replication cycles, this can result in lost information that could be critical for the individual to survive.

To solve this issue, telomeres are present in eukaryotes. Telomeres are short, repeating segments of DNA that are found at the end of each chromosome and do not contain any coding sequences. These telomeres are synthesized by telomerase , which is an enzyme that contains a short RNA template used to extend the length of the lagging strand. Primers are placed on the telomere where DNA polymerase III can attach to synthesize the final portion of DNA leftover on the lagging strand.

Telomere replication on the lagging strand is as follows:

• Telomerase attaches to the very end of the lagging strand, overhanging the unreplicated portion of DNA.
• Using its own RNA template, telomerase synthesizes the extending telomere, adding additional bases to the 3’ end of the lagging strand.
• Primase adds the primer on the telomere.
• DNA polymerase III binds to the primer and moves opposite of telomerase to complete the synthesis of the lagging strand.

Telomeres Affect Cell Age

Telomerase is most commonly active in cell types that divide rapidly, such as with embryonic cells, stem cells, sperm cells, and immune cells. In most other cell types, telomerase activity is turned off, and telomeres become shorter with each DNA replication. This means that cells have a limited number of times that they are able to divide via mitosis before signals are sent to prevent further divisions and DNA damage. As a result, cells have age.

Research has found that increasing telomere length can also increase the lifespan of the cell. While this offers a potential treatment to growth limiting cellular diseases, it also unfortunately assists cancer persistence and survival. Approximately 90% of cancer cells have mutated to turn on telomerase activity in cell types where it should be turned off. This causes another mechanism in which cancer cells can continue to divide without control and become immortalized. Research is ongoing to determine if/how deactivating telomerase activity can either slow or stop cancer progression.

DNA Repair and Damage

Incorrect replication.

DNA replication must be fast, but it must also be extremely accurate. DNA replication occurs trillions of times in a single human. Even if there was only a single mistake in each replication, that would add up to trillions of errors that could be detrimental to the individual’s life. So how are mistakes regulated?

The first way this is done is by DNA polymerase proofreading its own work. Each complimentary pair of nucleotides has a distinct shape. Therefore, when the wrong base is placed, the shape is different enough that DNA polymerase can recognize its own mistake. DNA polymerase can then cut out this wrong match and replace it with the correct base.

While DNA polymerase is able to proofread its own work, sometimes mistakes still goes amiss. Once DNA polymerase continues down the length of the strand, mismatch repair proteins are able to edit any additional mistakes. By using markers on the old strand of DNA, the mismatch repair proteins can distinguish sequence errors on the new strand. They then remove the mismatched nucleotide and replace it accordingly. With both DNA polymerase proofreading and the mismatch repair proteins correcting additional mistakes, there is roughly only one mistake for every 1 billion nucleotides synthesized.

Environmental Damage

Environmental factors- such a UV radiation, X-rays, and chemical exposure- can damage DNA. For example, UV radiation found in sunlight and tanning booths can create a thymine dimer where two thymine bases next to each other form a covalent bond. This thus creates a bump in the DNA strand that prevents DNA polymerase from synthesizing past this point. These circumstances can become detrimental, and systems must be put into place to repair damages such as this.

In the case of the UV radiation, eukaryotic cells have adapted a nucleotide excision repair system that is able to detect deformities in the shape of the DNA helix. At least 18 different proteins work together to remove this deformity, using the non-damaged strand as a template to repair the damaged strand. Prokaryotic cells have a simpler but similar nucleotide excision repair system that only requires three proteins. However, for UV radiation specifically, prokaryotes use an enzyme known as photolyase to detect this damage and make repairs.

DNA replication is a highly regulated molecular process where a single molecule of DNA is duplicated to result in two identical DNA molecules. As a semiconservative process, the double helix is broken down into two strands, where each strand serves as the template for the newly synthesized strand by matching complementary bases. Because DNA polymerase III can only synthesize the new strands from 5′-3′, this results in a leading strand that is continuously synthesized and a lagging strand that requires the use of Okazaki fragments. Meanwhile, because eukaryotes have linear DNA, telomeres are needed to ensure genetic information is not lost during replication. Because DNA is critical to life, research continues to better understand and treat diseases caused by mutations and damages in an individual’s DNA.

1. In double-stranded DNA, which nucleotide does adenine pair with?

2. What direction does DNA polymerase synthesize new DNA strands?

3. When the leading strand is being synthesized, what direction is the template strand?

4. When the lagging strand is being synthesized, what direction is the template strand?

5. DNA polymerase synthesizes new strands by matching complimentary base pairs from an external DNA template strand. There is not an external template for telomerase to use when synthesizing telomeres however. How does telomerase recognize what bases to add to the lagging strand and where to start?

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Bibliography

• Freeman, S., Quillin, K., Allison, L. A., Black, M., Podgorski, G., Taylor, E., & Carmichael, J. (2017). “Biological science (Sixth edition.).” Boston: Pearson Learning.
• Miesfeld, R. & McEvoy, M. (2017). “Biochemistry (Preliminary Edition).” New York: W.W. Norton & Company.
• Srinivas, N., Rachakonda, S., & Kumar, R. (2020). “Telomeres and Telomere Length: A General Overview.”  Cancers , 12  (3), 558.

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9.2 DNA Replication

Learning objectives.

• Explain the process of DNA replication
• Explain the importance of telomerase to DNA replication
• Describe mechanisms of DNA repair

When a cell divides, it is important that each daughter cell receives an identical copy of the DNA. This is accomplished by the process of DNA replication. The replication of DNA occurs during the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis.

The elucidation of the structure of the double helix provided a hint as to how DNA is copied. Recall that adenine nucleotides pair with thymine nucleotides, and cytosine with guanine. This means that the two strands are complementary to each other. For example, a strand of DNA with a nucleotide sequence of AGTCATGA will have a complementary strand with the sequence TCAGTACT ( Figure 9.8 ).

Because of the complementarity of the two strands, having one strand means that it is possible to recreate the other strand. This model for replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied ( Figure 9.9 ).

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. Each new double strand consists of one parental strand and one new daughter strand. This is known as semiconservative replication . When two DNA copies are formed, they have an identical sequence of nucleotide bases and are divided equally into two daughter cells.

DNA Replication in Eukaryotes

Because eukaryotic genomes are very complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination.

Recall that eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. How does the replication machinery know where on the DNA double helix to begin? It turns out that there are specific nucleotide sequences called origins of replication at which replication begins. Certain proteins bind to the origin of replication while an enzyme called helicase unwinds and opens up the DNA helix. As the DNA opens up, Y-shaped structures called replication forks are formed ( Figure 9.10 ). Two replication forks are formed at the origin of replication, and these get extended in both directions as replication proceeds. There are multiple origins of replication on the eukaryotic chromosome, such that replication can occur simultaneously from several places in the genome.

During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3' end of the template. Because DNA polymerase can only add new nucleotides at the end of a backbone, a primer sequence, which provides this starting point, is added with complementary RNA nucleotides. This primer is removed later, and the nucleotides are replaced with DNA nucleotides. One strand, which is complementary to the parental DNA strand, is synthesized continuously toward the replication fork so the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand . Because DNA polymerase can only synthesize DNA in a 5' to 3' direction, the other new strand is put together in short pieces called Okazaki fragments . The Okazaki fragments each require a primer made of RNA to start the synthesis. The strand with the Okazaki fragments is known as the lagging strand . As synthesis proceeds, an enzyme removes the RNA primer, which is then replaced with DNA nucleotides, and the gaps between fragments are sealed by an enzyme called DNA ligase .

The process of DNA replication can be summarized as follows:

• DNA unwinds at the origin of replication.
• New bases are added to the complementary parental strands. One new strand is made continuously, while the other strand is made in pieces.
• Primers are removed, new DNA nucleotides are put in place of the primers and the backbone is sealed by DNA ligase.

Visual Connection

You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

Telomere Replication

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached; however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres , which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase ( Figure 9.11 ) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn ( Figure 9.12 ) received the Nobel Prize for Medicine and Physiology in 2009. Later research using HeLa cells (obtained from Henrietta Lacks) confirmed that telomerase is present in human cells. And in 2001, researchers including Diane L. Wright found that telomerase is necessary for cells in human embryos to rapidly proliferate.

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

DNA Replication in Prokaryotes

Recall that the prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms.

DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes. Table 9.1 summarizes the differences between prokaryotic and eukaryotic replications.

Link to Learning

Click through a tutorial on DNA replication.

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then polymerization continues ( Figure 9.13 a ). Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base ( Figure 9.13 b ). In yet another type of repair, nucleotide excision repair , the DNA double strand is unwound and separated, the incorrect bases are removed along with a few bases on the 5' and 3' end, and these are replaced by copying the template with the help of DNA polymerase ( Figure 9.13 c ). Nucleotide excision repair is particularly important in correcting thymine dimers, which are primarily caused by ultraviolet light. In a thymine dimer, two thymine nucleotides adjacent to each other on one strand are covalently bonded to each other rather than their complementary bases. If the dimer is not removed and repaired it will lead to a mutation. Individuals with flaws in their nucleotide excision repair genes show extreme sensitivity to sunlight and develop skin cancers early in life.

Most mistakes are corrected; if they are not, they may result in a mutation —defined as a permanent change in the DNA sequence. Mutations in repair genes may lead to serious consequences like cancer.

• 1 Mariella Jaskelioff, et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature , 469 (2011):102–7.

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AP®︎/College Biology

Course: ap®︎/college biology   >   unit 6.

• Antiparallel structure of DNA strands
• Leading and lagging strands in DNA replication
• Speed and precision of DNA replication
• Semi-conservative replication
• Molecular mechanism of DNA replication

DNA structure and replication review

• Replication

DNA structure

Nucleotides, chargaff's rules, double helix, dna replication, the replication process, leading and lagging strands, example: determining a complementary strand, common mistakes and misconceptions.

• DNA replication is not the same as cell division. Replication occurs before cell division, during the S phase of the cell cycle. However, replication only concerns the production of new DNA strands, not of new cells.
• Some people think that in the leading strand, DNA is synthesized in the 5’ to 3’ direction, while in lagging strand, DNA is synthesized in the 3’ to 5’ direction. This is not the case. DNA polymerase only synthesizes DNA in the 5’ to 3’ direction only. The difference between the leading and lagging strands is that the leading strand is formed towards replication fork, while the lagging strand is formed away from replication fork.

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DNA Replication as a Semiconservative Process Essay

• To find inspiration for your paper and overcome writer’s block
• As a source of information (ensure proper referencing)
• As a template for you assignment

The process of DNA replication has been studied extensively as the pathway to understanding the processes of inheritance and the possible platform for addressing a range of health issues occurring as a result of DNA mutations.

However, the subject matter is still plagued by grey areas that require further analysis, the very properties of the process being one of the core issues of debating. Specifically, whether DNA replication can be deemed as semiconservative remains largely an unanswered question (Georgia Highlands College, n.d.). I believe that, despite the lack of certainty regarding the problem under analysis, it would be reasonable to believe that DNA replication is semiconservative since it is consistent with the fact that, during the reproduction process, DNS is separated into two bands.

The statement concerning DNA replication being a semiconservative process that leads to the development of two separate strands of DNA material has been supported by a vast range of evidence. Recent experiments point to the correctness of the semiconservative framework as the most legitimate theory that allows describing the process of DNA replication in the greatest detail possible (Georgia Highlands College, n.d.). In order to concede that the process of DNA replication is semiconservative, one should take a closer look at the outcomes of the experiment performed.

Since the test performed by Meselson and Stahl showed that the amount of the DNA material was equal in two daughter cells, yet the density thereof was different, the presence of semiconservative properties in DNA replication can be regarded as proven. By asserting that the observed tendency could be found in not only the strands of E.coli but also in other species, Meselson and Stahl made it evident that the DNA replication did, in fact, show semiconservative properties (Georgia Highlands College, n.d.). Thus, I insist that the existing evidence points to the DNA replication process being semiconservative.

The described outcomes of the experiment also lead to a vast range of conclusions concerning the nature and outcomes of DNA replications in different species. By defining DNA replication as semiconservative, the researchers made it evident that every double helix axis in the DNA structure built with the help of DNA polymerases leads to the creation of an entirely new strand that acts as complementary (Georgia Highlands College, n.d.).

It should also be borne in mind that the specified characteristic of the DNA structure makes it possible for the new strand, which is also known as the leading one, to emerge as a continuous piece, whereas the complementary one, or the lagging strand, occurs as a combination of smaller pieces (Georgia Highlands College, n.d.). By applying the notion of the DNA replication process being semiconservative, one can explain the observed changes within the DNA framework and provide the foundation for the further analysis of the subject matter.

Indeed, the results of the experiment described above cannot be deemed as consistent with the theory of dispersive replication, which has been offered as the alternative to the semiconservative framework. Using isotopes of nitrogen as the tools for labeling the DNA of the studied bacteria, Meselson and Stahl staged an experiment in the course of which the nature of the DNA replication process and the basis for its implementation were studied (Georgia Highlands College, n.d.). The semiconservative assumption made by the scientists implied that, in the process of replication, entirely new strands of DNA were produced with the help of the ones that were already present in the cells of the bacteria under analysis.

During the experiment, it was discovered that each of the nitrogenous bases presented in the DNA structure is only capable of connecting to its corresponding complementary partner. For adenine, the process of pairing occurs with thymine, whereas cytosine is connected to guanine in the process (Georgia Highlands College, n.d.). The resulting replication process, thus, takes place due to the combination of the helicase and DNA polymerase procedures (Georgia Highlands College, n.d.). Therefore, I strongly believe that the principle of DNA replication as the notion based on the semiconservative framework seems to be quite valid, given the vast amount of supportive evidence that has been collected.

Based on the outcomes of the Meselson–Stahl experiment, during which the DNA showed a strong propensity toward splitting into two distinct brands, the assumption that the DNA process is semiconservative can be regarded as confirmed. Even though it could be alleged that the current research has been erroneous and that the process of DNA replication may involve different processes and be based on an entirely different principle, the veracity of the identified statement is quite feeble. Thus, the stages of DNA replication can be seen as the semiconservative process. The presence of a synthesized strand along with the preexisting template one has proven to be the most sensible way of looking at the DNA replication stage.

Georgia Highlands College. (n.d.). Chapter 14 – DNA structure and function . Web.

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IvyPanda. (2021, June 2). DNA Replication as a Semiconservative Process. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/

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IvyPanda . 2021. "DNA Replication as a Semiconservative Process." June 2, 2021. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/.

1. IvyPanda . "DNA Replication as a Semiconservative Process." June 2, 2021. https://ivypanda.com/essays/dna-replication-as-a-semiconservative-process/.

Bibliography

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Chapter 14. DNA Replication

Chapter Outline

• 14.1 Historical Basis of Modern Understanding
• 14.2 Overview of DNA Replication
• 14.3 DNA Replication in Prokaryotes
• 14.4 DNA Replication in Eukaryotes
• 14.5 DNA Repair

Introduction

The three letters “DNA” have now become synonymous with crime solving, paternity testing, human identification, and genetic testing. DNA can be retrieved from hair, blood, or saliva. Each person’s DNA is unique, and it is possible to detect differences between individuals within a species on the basis of these unique features. DNA analysis has many practical applications beyond forensics. In humans, DNA testing is applied to numerous uses: tracing genealogy, identifying pathogens, archeological research, tracing disease outbreaks, and studying human migration patterns. In the medical field, DNA is used in diagnostics, new vaccine development, and cancer therapy. It is now possible to determine predisposition to diseases by looking at genes.

Each human cell has 23 pairs of chromosomes: one set of chromosomes is inherited from the mother and the other set is inherited from the father. There is also a mitochondrial genome, inherited exclusively from the mother, which can be involved in inherited genetic disorders. On each chromosome, there are thousands of genes, sequences of DNA that code for a functional product, that are responsible for determining the genotype and phenotype of the individual. The human haploid genome contains 3 billion base pairs and has between 20,000 and 25,000 functional genes.

In order for DNA to serve its role as the genetic material, all organisms must be able to faithfully copy the entire genome. This process,  DNA replication , is the precursor to all forms of cell division.

14.1 | Historical Basis of Modern Understanding

Learning Objectives

By the end of this section, you will be able to:

• Explain transformation of DNA.
• Describe the key experiments that helped identify that DNA is the genetic material.
• State and explain Chargaff’s rules

Modern understandings of DNA have evolved from the discovery of nucleic acids to the development of the double-helix model. In the 1860s, Friedrich Miescher ( Figure 14.2 ), a physician by profession, was the first person to isolate phosphate- rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.

A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation , a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule; as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle ( Figure 11.3 ). These experiments are now famously known as Griffith’s transformation experiments.

Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.

Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35 S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32 P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.

Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant (the liquid above the pellet). In the tube that contained phage labeled with 35 S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32 P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins ( Figure 14.4 ).

Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. These are also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model, discussed in Chapter 5.

14.2 | Overview of DNA Replication

• Explain how the structure of DNA reveals the replication process.
• Describe the Meselson and Stahl experiments.

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested ( Figure 14.5 ): conservative, semi-conservative, and dispersive.

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed. Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N) that gets incorporated into nitrogenous bases, and eventually into the DNA ( Figure 14.6 ).

The  E. coli  culture was then shifted into medium containing  14 N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in  14 N and spun again. During the density graditent centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in  15 N will band at a higher density position than that grown in  14 N. Meselson and Stahl noted that after one generation of growth in  14 N after they had been shifted from  15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in  15 N and  14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in  14 N formed two bands: one DNA band was at the intermediate position, between  15 N and  14 N and the other corresponded to the band of  14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

14.3 | DNA Replication in Prokaryotes

• Explain the process of DNA replication in prokaryotes.
• Discuss the role of different enzymes and proteins in supporting this process.

DNA replication has been extremely well studied in prokaryotes primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs without many mistakes.

DNA replication employs a large number of proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one by one to the growing DNA chain that are complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleotides that have three phosphates attached to them, similar to ATP which has three phosphate groups attached. When the bond between the phosphates is broken, the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.

How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), it is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi- directionally as replication proceeds.  Single-strand binding proteins  coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix. DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand can be only extended in this direction). It also requires a free 3′- OH group to which it can add nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3′-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3′-OH end. Another enzyme, RNA primase , synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are complementary to the template strand ( Figure 14.7 ).

The replication fork moves at the rate of 1000 nucleotides per second. DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the replication fork. As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′ direction and the other is oriented in the 3′ to 5′ direction. One strand, which is complementary to the 3′ to 5′ parental DNA strand, is synthesized continuously towards the replication fork because the polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand . The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments , each requiring a primer to start the synthesis. Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand .

Concept check

You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated?

The leading strand can be extended by one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to 3′. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, and the gaps are filled in by deoxyribonucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′ phosphate end of the other fragment.

Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division. The process of DNA replication can be summarized as follows:

• DNA unwinds at the origin of replication.
• Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
• Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
• Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
• Primase synthesizes RNA primers complementary to the DNA strand.
• DNA polymerase starts adding nucleotides to the 3′-OH end of the primer.
• Elongation of both the lagging and the leading strand continues.
• RNA primers are removed by exonuclease activity.
• Gaps are filled by DNA pol by adding dNTPs.
• The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.

Table 11.1 Prokaryotic DNA replication: enzymes and their functions.

14.4 | DNA Replication in Eukaryotes

• Discuss the similarities and differences between DNA replication in eukaryotes and prokaryotes.
• State the role of telomerase in DNA replication.

14.4.1 Prokaryote vs. Eukaryote Replication

Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on the eukaryotic chromosome; humans can have up to 100,000 origins of replication. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as Autonomously Replicating Sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E. coli.

The number of DNA polymerases in eukaryotes is much more than prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as pol α, pol β, pol γ, pol δ, and pol ε.

The essential steps of replication are the same as in prokaryotes. Before replication can start, the DNA has to be made available as template. Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Other proteins are then recruited to start the replication process ( Table 11.2 ).

A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis. While the leading strand is continuously synthesized by the enzyme pol δ, the lagging strand is synthesized by pol ε. A sliding clamp protein known as PCNA (Proliferating Cell Nuclear Antigen) holds the DNA pol in place so that it does not slide off the DNA. RNase H removes the RNA primer, which is then replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined together after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond.

Table 11.2 Differences between Prokaryotic and Eukaryotic Replication

14.4.2 Telomere Replication

Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5′ to 3′ direction. In the leading strand, synthesis continues until the end of the chromosome is reached. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and over time these ends may get progressively shorter as cells continue to divide.

The ends of the linear chromosomes are known as telomeres , which have repetitive sequences that code for no particular gene. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase ( Figure 14.8 ) helped in the understanding of how chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3′ end of the DNA strand. Once the 3′ end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For her discovery of telomerase and its action, Elizabeth Blackburn ( Figure 14.9 ) received the Nobel Prize for Medicine and Physiology in 2009.

Telomerase and Aging

Cells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older.

In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine [1] . Telomerase-deficient mice were used in these studies; these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.

14.5 | DNA Repair

• Discuss the different types of mutations in DNA.
• Explain DNA repair mechanisms.

DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations; in other cases, repair enzymes are themselves mutated or defective.

Most of the mistakes during DNA replication are promptly corrected by DNA polymerase by proofreading the base that has been just added ( Figure 14.10 ). In proofreading , the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.

Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair ( Figure 14.11 ). The enzymes recognize the incorrectly added nucleotide and excise it; this is then replaced by the correct base. If this remains uncorrected, it may lead to more permanent damage. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli , after replication, the nitrogenous base adenine acquires a methyl group; the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA polymerase is able to remove the wrongly incorporated bases from the newly synthesized, non- methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.

In another type of repair mechanism, nucleotide excision repair , enzymes replace incorrect bases by making a cut on both the 3′ and 5′ ends of the incorrect base ( Figure 14. 12 ). The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa ( Figure 14. 13 ). Affected individuals have skin that is highly sensitive to UV rays from the sun. When individuals are exposed to UV, pyrimidine dimers, especially those of thymine, are formed; people with xeroderma pigmentosa are not able to repair the damage. These are not repaired because of a defect in the nucleotide excision repair enzymes, whereas in normal individuals, the thymine dimers are excised and the defect is corrected. The thymine dimers distort the structure of the DNA double helix, and this may cause problems during DNA replication. People with xeroderma pigmentosa may have a higher risk of contracting skin cancer than those who dont have the condition.

Errors during DNA replication are not the only reason why mutations arise in DNA. Mutations , variations in the nucleotide sequence of a genome, can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of natural reactions taking place within the body.

Mutations may have a wide range of effects. Some mutations are not expressed; these are known as silent mutations . Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These can be of two types, either transitions or transversions . Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa; for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Mutations can also be the result of the addition of a base, known as an insertion , or the removal of a base, also known as deletion . Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome; this is also known as translocation . Some of these mutation types are shown in Figure 14.14.

Mutations in repair genes have been known to cause cancer. Many mutated repair genes have been implicated in certain forms of pancreatic cancer, colon cancer, and colorectal cancer. Mutations can affect either somatic cells or germ cells. If many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. If a mutation takes place in germ cells, the mutation will be passed on to the next generation, as in the case of hemophilia and xeroderma pigmentosa.

• Jaskelioff et al., “Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice,” Nature 469 (2011): 102-7. ↵

Introduction to Molecular and Cell Biology Copyright © 2020 by Katherine R. Mattaini is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Microbe Notes

DNA Replication: Enzymes, Mechanism, Steps, Applications

DNA replication is the process of producing two identical copies of DNA from one original DNA molecule.

• DNA is made up of millions of nucleotides, which are composed of deoxyribose sugar, with phosphate and a base. 
• The complementary pairing of these bases keeps the double strands intact. So, to make two copies of one DNA, these hydrogen bonds in between the bases should be broken to begin replication.
• DNA replication is semi-conservative, meaning that each strand in the DNA acts as a template for the synthesis of a new complementary strand. Semi conservative because once DNA molecule is synthesized it has one strand from the parent and the other strand is a newly formed strand. 
• DNA replication starts by taking one DNA molecule and giving two daughter molecules, with each newly synthesized molecule containing one new and one old strand. 
• DNA replication simply is the process by which a DNA makes a copy of itself. Though easy as it may sound it’s a complex process happening inside of our cells, and many enzymes, proteins, and metal ions should work coherently to make this process happen.

Table of Contents

Interesting Science Videos

Mechanism of DNA Replication

Summary : DNA replication takes place in three major steps.

• Opening of the double-stranded helical structure of DNA and separation of the strands
• Priming of the template strands
• Assembly of the newly formed DNA segments.
• During the separation of DNA, the two strands uncoil at a specific site known as the origin . With the involvement of several enzymes and proteins, they prepare (prime) the strands for duplication.
• At the end of the process, DNA polymerase enzyme starts to organize the assembly of the new DNA strands.
• These are the general steps of DNA replication for all cells but they may vary specifically, depending on the organism and cell type.
• Enzymes play a major role in DNA replication because they catalyze several important stages of the entire process.
• DNA replication is one of the most essential mechanisms of a cell’s function and therefore intensive research has been done to understand its processes.
• The mechanism of DNA replication is well understood in Escherichia coli, which is also similar to that in eukaryotic cells.
• In E.coli, DNA replication is initiated at the oriClocus (oriC), to which DnaA protein binds while hydrolyzing of ATP takes place.

Enzymes and Proteins Used in DNA Replication

• A nuclease is an enzyme that can cleave the phosphodiester bonds present in between the nucleotides.
• On the basis where they cleave, they are characterized as Exo and endonucleases.
• Exonucleases cleave nucleotides from their respective ends. Corresponding to this fact, these exonucleases show activity from both directions 5′ to 3′ and 3′ to 5′.
• Endonucleases act on the region in the middle of the targeted nucleotide. They are also endonucleases that are selective to which molecule they cleave and are sub-divided as DNase for DNA for cleaving and RNase for RNA cleaving. Additionally, recently discovered nucleases are also being used for gene editing such as Cas9 in the CRISPR genome editing technique.
• Restrictive endonuclease or restriction enzymes are the ones that cleave DNA into fragments at or near the specific recognition sites within the molecule known as restriction sites.
• To cleave the DNA, restriction endonuclease makes two incisions, once through each sugar-phosphate backbone of the DNA double helix. These endonucleases recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA.
• This specific sequence is usually 4 – 8 bases and is present in the recognition site.
• DNA Polymerase

• DNA polymerases are the enzyme that is responsible for adding new nucleotides and synthesizing a new strand of DNA by taking the old fragmented strand as a template.
• DNA Polymerases also possess exonuclease activity, that cuts incorrectly added nucleotides, and allows the DNA replication to happen without errors.
• DNA Polymerase is of many types and functions based on the cell they are found in. 
• In prokaryotic cells, there are three DNA polymerases: DNA Polymerase Ι, DNA Polymerase ΙΙ and DNA Polymerase ΙΙΙ.
• DNA polymerase Ι is a repair polymerase with 5′ to 3′ and 3′ to 5′ exonuclease activity. It is involved in the processing of Okazaki fragments during lagging strand synthesis.
• DNA polymerase ΙΙ has 3′ to 5′ exonuclease activity and participated in DNA repair with 5′ to 3′ polymerase activity.
• DNA polymerase ΙΙΙ is the primary enzyme involved in the DNA replication of E.coli . It has 3′ to 5′ exonuclease activity and 5′ to 3′ polymerase activity.
• In eukaryotic cells, there are five DNA polymerases: DNA Polymerase α, β, γ, δ and ε 
• DNA polymerase α is a repair polymerase, with 3′ to 5′ exonucleases activities and 5′ to 3′ polymerase activities.
• DNA Polymerase β is a repair polymerase.
• DNA Polymerase γ shows polymerase activity 5′ to 3′ and exonucleases activity 3′ to 5′, it is involved in Mitochondrial DNA replication 
• DNA Polymerase δ shows 3′ to 5′ exonuclease activity and 5′ to 3′ polymerase activity. This enzyme is involved in lagging strand synthesis.
• DNA Polymerase ε shows 3′ to 5′ and 5′ to 3′ exonucleases activities. This enzyme not only repairs but also synthesizes the leading strand efficiently in a 5′ to 3′ direction. It is the prime enzyme involved in DNA replication.

• DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.
• This enzyme joins the 3′ hydroxyl group of one nucleotide with the 5′ phosphate end of another nucleotide at an expense of ATP.
• DNA helicase
• DNA helicase is a motor protein that moves directionally along a nucleic acid phosphodiester backbone, separating two nucleotides of DNA molecule.
• They separate double-stranded DNA molecules into single strands allowing each strand to be copied.
• During DNA replication, this DNA helicase unwinds DNA at the origin, a site where the replication is to be initiated.
• DNA helicase continues to unwind the double helix of DNA and thus forms a structure called replication fork, named after the forked appearance of two strands of DNA when unzipped apart. 
• It is an energy-driven process as it involves the breaking of Hydrogen bonds between annealed nucleotide bases. 
• DNA primase
• Primase is an enzyme that is capable to synthesize short stretches of RNA sequences known as a primer.
• Primers are an integral part of DNA replication. These primers serve as an initiating site for the addition of nucleotides by DNA polymerase. 
• DNA polymerase can only add nucleotide at pre-existing 3′ Hydroxyl group which is thus provided by the primers.
• As we can see that primers are short stretches of RNA, but replication is of DNA, so therefore after elongation of the chains of nucleotides, these primers are replaced by DNA. 
• DNA topoisomerase
• DNA topoisomerase is a class of enzymes that release helical tension during transcription and replication by creating transient nicks within the phosphate backbone on one or both strands of the DNA.
• This tension is aroused when the DNA molecule unwinds due to helicase activity and forms a replication fork. The progress of the replication fork generates supercoils, making it hard for other machinery involved to access the DNA molecule.
• Class Ι DNA topoisomerase makes a single-stranded break to relax the helix and progress the process.
• Class ΙΙ DNA topoisomerase break both the strands of DNA helix, this class of topoisomerases is also very important during the cell cycle for the condensation of chromosomes.
• Single strand binding proteins
• The single-strand binding (SSB) protein are DNA binding proteins, that binds to single-stranded DNA to facilitate DNA replication.
• SSB proteins prevent the hardening of strands during DNA replication. It also protects strands from nuclease degradation and prevents the rewinding of DNA. 
• These proteins destabilize helical duplexes so that DNA polymerase can hold onto the DNA during DNA replication, recombination, and repair.
• It also removes unwanted secondary structures on strands for easy access of the strands to the machinery involved in DNA replication.
• Thus, SSB proteins stabilize the single-stranded DNA structure that is important for genomic progression.

So, in summary here are the list of 7 enzymes and proteins used in DNA Replication:

Steps in DNA Replication

Step 1: formation of replication fork.

• Before DNA can replicate, this double-stranded molecule must unwind into two single strands to initiate the replication process.
• DNA unwinds when the complementary base pairing between the double-stranded is broken, and the site to initiate this unwinding is denoted by specific regions (Adenine and Thymine rich).
• These specific coding regions are referred to as Origin of Replication (Ori) and thus the replication process begins.
• These origins are targeted by initiator proteins, which go on to recruit more proteins that can help the replication process by forming a replication fork around the Ori. 
• Within this replication protein complex is an enzyme DNA helicase, which starts to unwind the DNA from its Ori and exposes two strands resembling a Y-like structure referred to as replication fork. 
• The activity of helicase causes topological stress to the un-winded strand forming supercoiled DNA, this stress is relieved by Topoisomerase by negative supercoiling.
• The replication fork is bidirectional; one strand is oriented to 5′ to 3′ direction (leading strand) and the other strand is oriented to 3′ to 5′ direction (lagging strand) but the addition of nucleotide progress only in 5′ to 3′ direction.
• The formation of a replication fork exposing two single-stranded strands marks the beginning of Initiation.

Step 2: Initiation

• One strand runs from 5′ to 3′ direction towards the replication fork and is referred to as leading strand and the other strand runs from 3′ to 5′ away from the replication fork and is referred to as lagging strands. 
• To this exposed single-stranded DNA, SSB proteins are adhered to prevent recoiling of DNA and to stabilize it.
• After which another enzyme DNA primase comes into action to synthesize a short stretch of RNA primer, which provides a free 3′ hydroxyl group for DNA polymerase can now add nucleotides and extend the new chain of nucleotides.

Step 3: Elongation

• Now that primer is added to unzipped two single-stranded DNA, these strands now act as a template for synthesizing new DNAs.
• The enzyme DNA polymerase synthesizes new nucleotide to match the template and add on to the free 3′ hydroxyl group provided by the primer in each single-stranded DNA.
• The leading strand runs from 5′ to 3′ so the addition of nucleotides by DNA polymerase happens from 5′ to 3′ direction. As the replication fork progresses the addition of nucleotide is continuous thus only requiring the primer once.
• However, lagging strands is antiparallel and run from the 5′ to 3′ direction, the continuous addition of nucleotides is not possible as the replication fork progresses, DNA polymerase cannot add complementary nucleotides to the 5′ end. Therefore, multiple primers are required.
• Due to this phenomenon, the DNA nucleotides synthesis from lagging strands occurs in fragments. These fragments are termed Okazaki fragments.
• Hence, the leading strand using only one primer synthesizes nucleotides continuously, while the lagging strand uses multiple primers and thus synthesizes nucleotides discontinuously. 

Step 4: Termination

• RNA primers of both leading and lagging strands are cleaved out or degraded by exonucleases activity of DNA polymerase, and the nicks or gaps so formed are filled with DNA and sealed by the enzyme DNA ligase.
• DNA polymerase also shows proofreading activity and check, remove and replace any errors.
• Interestingly, in eukaryotic organisms having linear DNA, when RNA primer at 5′ end of daughter strand is removed, there is not a preceding 3′ OH such that DNA polymerase can use it to replace with DNA.
• So, at the 5′ end of daughter strands, there is a gap (missing DNA). This missing DNA can cause a loss of information contained in that region. This gap must be filled before the next round of replication.
• For solving this end replication problem, researchers have found that linear ends of DNA called telomere are used which contain specific G: C rich repeats. These sequences are known as telomere sequences.
• These telomere sequences do not code anything but are essential to fill in the gap in the daughter strand and maintain the integrity of DNA.
• Eventually, the replication forks terminate at terminating recognizing sequences (ter).
• The ter sequences are of around 23 base pairs which facilitate as the binding sites for TUS protein.
• This ter- TUS complex arrest replication fork and terminate.

So, in summary, these are the 4 steps of DNA Replication:

• Formation of Replication Fork
• Termination

Okazaki Fragments

• The two DNA strands run in opposite or antiparallel directions, and therefore to continuously synthesize the two new strands at the replication fork requires that one strand is synthesized in the 5’to3′ direction while the other is synthesized in the opposite direction, 3’to 5′.
• However, DNA polymerase can only catalyze the polymerization of the dNTPs only in the 5’to 3’direction.
• This means that the other opposite new strand is synthesized differently. But how?
• By the joining of discontinuous small pieces of DNA that are synthesized backward from the direction of movements of the replication fork. These small pieces or fragments of the new DNA strand are known as the Okasaki Fragments.
• The Okasaki fragments are then joined by the action of DNA ligase, which forms an intact new DNA strand known as the lagging strand.
• The lagging phase is not synthesized by the primer that initiates the synthesis of the leading strand.
• Instead, a short fragment of RNA serves as a primer (RNA primer) for the initiation of replication of the lagging strand.
• RNA primers are formed during the synthesis of RNA which is initiated de novo, and an enzyme known as primase synthesizes these short fragments of RNA, which are 3-10 nucleotides long and complementary to the lagging strand template at the replication fork.
• The Okazaki fragments are then synthesized by the extension of the RNA primers by DNA polymerase.
• However, the newly synthesized lagging strand is that it contains an RNA-DNA joint, defining the critical role of RNA in DNA replication.

Replication Fork Formation and its function

• The replication fork is the site of active DNA synthesis, where the DNA helix unwinds and single strands of the DNA replicates.
• Several sites of origin represent the replication forks.
• The replication fork is formed during DNA strand unwinding by the helicase enzyme which exposes the origin of replication. A short RNA primer is synthesized by primase and elongation done by DNA polymerase.
• The replication fork moves in the direction of the new strand synthesis. The new DNA strands are synthesized in two orientations, i.e. 5′ to 3′ direction which is the leading strand, and the 3′ to 5′ orientation which is the lagging strand.
• The two sides of the new DNA strand (leading and lagging strand) are replicated in two opposite directions from the replication fork.
• Therefore the replication fork is bi-directional.

Leading Strand

• The leading strand is the new DNA strand that is continuously synthesized by the DNA polymerase enzyme.
• It is the simplest strand that is synthesized during replication.
• The synthesis starts after the DNA strand has unzipped and separated. This generates a short piece of RNA known as a primer , by the DNA primase enzyme.
• The primer binds to the 5′ end (start) of the strand, thus initiating the synthesize of the new strand (leading strand).
• The synthesis of the leading strand is a continuous process.

The Lagging Strand

• This is the template strand (3′ to 5′) that is synthesized in a discontinuous manner by RNA primers.
• During the synthesis of the leading strand, it exposes small, short strands, or templates that are then used for the synthesis of the Okasaki fragments.
• The Okasaki fragments synthesize the lagging strand by the activity of DNA polymerase which adds the pieces of DNA (the Okasaki fragments) to the strand between the primers.
• The formation of the lagging strand is a discontinuous process because the newly formed strand (lagging strand) is the fragmentation of short DNA strands.

Applications of DNA Replication

• DNA replication makes the transfer of genetic information from one generation to another possible.
• It is an important phenomenon happening inside our cells, that allows the body to maintain homeostasis and integrity of the body.
• With the available information about DNA replication, scientific communities today have a proper idea of genome sequencing which has now been applied in different expertise ranging from clinical diagnosis to possible treatment of genetic diseases.
• DNA replication has made sequencing of whole human genome sequencing possible.
• Cloning of genes has also been possible by DNA replication.
• Enzymes involved in DNA replication have now been greatly studied due to their wider applications. The recent breakthrough Cas9/ CRISPR technology where nucleases are used to cleave the desired portion of DNA and replace it with required nucleotides is the prime example of how we can use these enzymes and make potential advancements in them thus broadening and exploring their uses.
• Polymerase Chain Reaction uses DNA polymerases to repeatedly replicate DNA in-vitro and has numerous applications in diagnosis, sequencing, and recombinant DNA technology.
• The formation of complementary DNA (cDNA) can also be considered as an example of a wider application of the enzymes involved in DNA replication.
• There are various applications of DNA replication, we can even consider that if there is any technique involving genes, some way or the other DNA replication is applied.

DNA Replication Stress

During DNA replication, the process and the DNA genome undergoes various stress arising from the mechanism. these stresses an result in stalled replication and stalled replication fork formation. Several events contribute to these stresses, including;

• Unusual DNA structure
• Mismatched ribonucleotides
• Tensions arising from concurrent mechanisms of replication and transcription
• Inadequate availability of important replication factors
• Fragile sites on the replicating DNA strand
• Overexpression or constitutive activation of oncogenes
• Inaccessible chromatins

Kinase regulatory proteins such as ATM ( ATM serine/threonine kinase ) and ATP are proteins that assist in alleviating replication stress. These proteins get recruited and activated by DNA damages .

Stalled replication forks may collapse if the regulatory proteins do not stabilize, and if and when this happens, initiation of repairing mechanisms to reassembling of the replication fork takes place. this helps to amend damages the damaged ends of DNA.

Similarities between Prokaryotic and Eukaryotic DNA Replication

• The unwinding mechanism of DNA before replication is initiated is the same for both Prokaryotes and eukaryotes.
• In both organisms, the DNA polymerase enzyme coordinated the synthesis of new DNA strands.
• Additionally, both organisms use the semi-conservative replication pattern, making the leading and lagging strands in different directions. Okasaki fragments make the lagging strand.
• Lastly, both organisms initiate DNA replication using a short RNA primer.

Eukaryotic vs. Prokaryotic DNA Replication (11 Major Differences)

• Weiguo Cao, “DNA Ligases: Structure, Function, and Mechanism”, Current Organic Chemistry 2002; 6(9). https://doi.org/10.2174/1385272023373950
• https://www.nature.com/scitable/definition/helicase-307/
• https://proteinswebteam.github.io/interpro-blog/potm/2006_1/Page2.htm
• https://www.yourgenome.org/facts/what-is-dna-replication
• https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/replication/a/molecular-mechanism-of-dna-replication
• https://teachmephysiology.com/biochemistry/cell-growth-death/dna-replication/
• https://courses.lumenlearning.com/wm-biology1/chapter/reading-major-enzymes/
• https://geneticeducation.co.in/single-stranded-binding-protein-ssb-structure-and-function/
• https://www.thoughtco.com/dna-replication-3981005
• https://byjus.com/biology/difference-between-prokaryotic-and-eukaryotic-replication/
• https://www.vedantu.com/biology/difference-between-prokaryotic-and-eukaryotic-dna-replication
• https://www.majordifferences.com/2013/03/difference-between-prokaryotic-and.html

About Author

Rajat Thapa

2 thoughts on “DNA Replication: Enzymes, Mechanism, Steps, Applications”

hello i got a doubt about leading and lagging strand directions. 5` to 3` should be leading strand and 3` to 5` should be a lagging strand. because the polymerase activity goes in 5` to 3` direction so the strand is continuous without any breaks so it is called leading strand and the strand which is in opposite direction to the direction of polymerase is disturbed with breaks hence it is called lagging strand. but you reversed it so please check it and please correct it.

Hello, Yes, you are correct. There has been slight mistakes on the note. We have corrected the note and updated it. Thank you so much for your finding.

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Mitochondrial DNA replication in mammalian cells: overview of the pathway

Maria falkenberg.

Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, 405 30 Gothenburg, Sweden

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome and a dedicated DNA replication machinery is required for its maintenance. Many disease-causing mutations affect mitochondrial replication factors and a detailed understanding of the replication process may help to explain the pathogenic mechanisms underlying a number of mitochondrial diseases. We here give a brief overview of DNA replication in mammalian mitochondria, describing our current understanding of this process and some unanswered questions remaining.

Introduction

Mitochondrial DNA (mtDNA) is a double-stranded molecule of 16.6 kb ( Figure 1 , lower panel). The two strands of mtDNA differ in their base composition, with one being rich in guanines, making it possible to separate a heavy (H) and a light (L) strand by density centrifugation in alkaline CsCl 2 gradients [ 1 ]. The mtDNA contains one longer noncoding region (NCR) also referred to as the control region. In the NCR, there are promoters for polycistronic transcription, one for each mtDNA strand; the light strand promoter (LSP) and the heavy strand promoter (HSP). The NCR also harbors the origin for H-strand DNA replication (O H ). A second origin for L-strand DNA replication (O L ) is located outside the NCR, within a tRNA cluster approximately 11,000 bp downstream of O H .

The genome encodes for 13 mRNA (green), 22 tRNA (violet), and 2 rRNA (pale blue) molecules. There is also a major noncoding region (NCR), which is shown enlarged at the top. The major NCR contains the heavy strand promoter (HSP), the light strand promoter (LSP), three conserved sequence boxes (CSB1-3, orange), the H-strand origin of replication (O H ), and the termination-associated sequence (TAS, yellow). The triple-stranded displacement-loop (D-loop) structure is formed by premature termination of nascent H-strand DNA synthesis at TAS. The short H-strand replication product formed in this manner is termed 7S DNA. A minor NCR, located approximately 11,000 bp downstream of O H , contains the L-strand origin of replication (O L ).

mtDNA replication factors

Mammalian mtDNA is replicated by proteins distinct from those used for nuclear DNA replication and many are related to replication factors identified in bacteriophages [ 2 ]. DNA polymerase γ (POLγ) is the replicative polymerase in mitochondria. In human cells, POLγ is a heterotrimer with one catalytic subunit (POLγA) and two accessory subunits (POLγB) [ 3–5 ]. Mouse knockouts for POLγA and POLγB have revealed that both factors are essential for embryonic development [ 6 , 7 ]. At least four additional polymerases (PrimPol, DNA polymerase β, DNA polymerase θ, and DNA polymerase ζ) have been reported to play a role mitochondria [ 8–11 ]. These polymerases are not essential for mtDNA maintenance and none of them can substitute for POLγ. Most likely they are involved in mtDNA repair, but the exact function of these additional polymerases in mtDNA maintenance needs to be further elucidated (reviewed in [ 12 ]).

POLγA belongs to the family A DNA polymerases and contains a 3′–5′ exonuclease domain that acts to proofread the newly synthesized DNA strand [ 3 ]. POLγ is a highly accurate DNA polymerase with a frequency of misincorporation lower than 1 × 10 −6 [ 13 ]. The accessory POLγB subunit enhances interactions with the DNA template and increases both the catalytic activity and the processivity of POLγA [ 14–16 ]. POLγ cannot use double-stranded DNA as a template and a DNA helicase is therefore required at the mitochondrial replication fork [ 17 ]. The DNA helicase TWINKLE is homologous to the T7 phage gene 4 protein [ 18 ] and during mtDNA replication, TWINKLE travels in front of POLγ, unwinding the double-stranded DNA template. TWINKLE forms a hexamer and requires a fork structure (a single-stranded 5′-DNA loading site and a short 3′-tail) to load and initiate unwinding [ 18–20 ]. Mitochondrial single-stranded DNA-binding protein (mtSSB) binds to the formed ssDNA, protects it against nucleases, and prevents secondary structure formation [ 21 , 22 ]. mtSSB enhances mtDNA synthesis by stimulating TWINKLE’s helicase activity as well as increasing the processivity of POLγ [ 17 , 19 , 23 ].

The mode of mtDNA replication

A model for mtDNA replication was presented already in 1972 by Vinograd and co-workers ( Figure 2 ) [ 24 ]. According their strand displacement model, DNA synthesis is continuous on both the H- and L-strand [ 25 ]. There is a dedicated origin for each strand, O H and O L . First, replication is initiated at O H and DNA synthesis then proceeds to produce a new H-strand. During the initial phase, there is no simultaneous L-strand synthesis and mtSSB covers the displaced, parental H-strand [ 26 ]. By binding to single-stranded DNA, mtSSB prevents the mitochondrial RNA polymerase (POLRMT) from initiating random RNA synthesis on the displaced strand [ 27 ]. When the replication fork has progressed about two-thirds of the genome, it passes the second origin of replication, O L . When exposed in its single-stranded conformation, the parental H-strand at O L folds into a stem–loop structure [ 28 ]. The stem efficiently blocks mtSSB from binding and a short stretch of single-stranded DNA in the loop region therefore remains accessible, allowing POLRMT to initiate RNA synthesis [ 26 , 29 ]. POLRMT is not processive on a single-stranded DNA templates [ 27 ]. Already after about 25 nt, it is replaced by POLγ and L-strand DNA synthesis is initiated [ 30 ]. From this point, H- and L-strand synthesis proceeds continuously until the two strands have reached full circle. Replication of the two strands is linked, since H-strand synthesis is required for initiation of L-strand synthesis. The structure and the sequence requirement for mammalian O L has been studied both in vivo and in vitro , demonstrating that a functional human O L must include a stable double-stranded stem region with pyrimidine-rich template strand and a single-stranded loop of at least 10 nt [ 31 ].

Mitochondrial DNA replication is initiated at O H and proceeds unidirectionally to produce the full-length nascent H-strand. mtSSB binds and protects the exposed, parental H-strand. When the replisome passes O L , a stem–loop structure is formed that blocks mtSSB binding, presenting a single-stranded loop-region from which POLRMT can initiate primer synthesis. The transition to L-strand DNA synthesis takes place after about 25 nt, when POLγ replaces POLRMT at the 3′-end of the primer. Synthesis of the two strands proceeds in a continuous manner until two full, double-stranded DNA molecules have been formed.

It should be noted that some aspects of the strand-displacement model have been questioned. For instance, studies have suggested that processed RNA molecules hybridize to the single-stranded H-strand and function as a provisional lagging strand, which is replaced by DNA during later stages of mtDNA replication [ 32 ]. This so-called RITOLS model implicates that processed transcripts are successively hybridized to the paternal H-strand as the replication fork advances, but the enzymatic machinery required for this process has not been identified [ 33 ]. Arguing against the RITOLS model, single-stranded DNA binding proteins are used to stabilize single-stranded DNA intermediates during DNA replication in all three major branches of life. In mitochondria, there are at least 500 mtSSB tetramers available per mtDNA molecule. Since each tetramer binds 59 nt, the levels of mtSSB are sufficient to cover the entire parental H-strand during mtDNA synthesis [ 26 , 34 ]. In addition, strand-specific chromatin immunoprecipitation has revealed that mtSSB exclusively covers the parental H-strand during mtDNA replication in vivo . The occupancy profile displays a distinct pattern, with the highest levels of mtSSB close to OriH, followed by a gradual decline toward OriL. The pattern is thus as would be predicted if mtSSB functions to stabilize the single-stranded, paternal H-strand during strand-displacement DNA replication [ 26 ]. Yet another problem for the RITOLS model is the presence of RNASEH1 in mitochondria. This enzyme efficiently removes RNA–DNA hybrids, an activity that is difficult to reconcile with processed RNA molecules stably binding to long stretches of single-stranded DNA during mtDNA replication [ 35 ].

Finally, there have been reports suggesting that under certain conditions, strand-coupled replication may function as a backup replication mode in mammalian mitochondria [ 36 , 37 ]. The molecular mechanisms underlying this type of replication have not been elucidated.

Curiously, not all replication events initiated at O H continue to full circle. Instead, 95% are terminated already after about 650 nt at the termination associated sequences (TAS) [ 38 , 39 ]. The short DNA fragment formed in this way, 7S DNA, remains bound to the parental L-strand, while the parental H-strand is displaced ( Figure 1 , top panel). As a result, a triple-stranded displacement loop structure, a D-loop, is formed. The functional importance of the D-loop structure is unclear and how replication is terminated at TAS is also not known [ 40 , 41 ]. It appears however that termination at TAS is a regulated event, providing a switch between abortive and genome length mtDNA replication [ 42 ]. In support of this notion, in vivo occupation analysis revealed that POLγ under normal conditions stalls at the 3′-end of the D-loop, whereas TWINKLE occupancy is low in this region. When mtDNA is depleted, the situation changes and TWINKLE occupancy increases and at the same time 7S DNA levels are decreased. These data have been interpreted as evidence for TWINKLE reloading in response to increased demand for mtDNA replication [ 42 ]. Binding of the helicase to the 3′-end of 7S DNA would allow the stalled POLγ to continue replication of 7S DNA to full circle. The model receives support from mouse genetic experiments, demonstrating that TWINKLE is important for mtDNA copy number control. Increased or decreased levels of TWINKLE correlate nicely with mtDNA levels [ 43–46 ]. It is thus possible that mtDNA replication is regulated at the level of pretermination rather than initiation. The switch may fine-tune the mtDNA copy number in response to cellular demands.

Interestingly, two closely related 15 nt and evolutionary conserved palindromic sequence motifs (ATGN 9 CAT) are located on each side of the D-loop region. One motif is located just upstream of the 5′-end of the 7S DNA, where it forms a part of conserved sequence box 1, CSB1. The second motif, core-TAS, is located within the TAS region, just downstream of the 3′-end of 7S DNA ( Figure 1 , top panel) [ 42 ]. The physiological role of these motifs remains unclear, but sequence-specific DNA binding proteins often recognize and bind palindromic sequences. In support of this notion, there are published in organello footprints located to the TAS region [ 47 ], but despite substantial efforts in different laboratories, a TAS-binding protein has so far not been identified. It is possible that the proteins binding to CSB1, core-TAS, and other regions within TAS are difficult to purify by traditional methods. Perhaps the missing protein is membrane bound and difficult to retain in solution during chromatography. Alternatively, binding could be a regulated event, requiring precise redox conditions or nucleotide concentrations. Finally, it cannot be excluded that secondary structures in mtDNA may play a role, e.g. stem–loops or G-quadruplexes, which could also contribute to the observed in vivo DNA footprints.

Initiation of mtDNA replication at O H

We know that POLRMT forms the primers necessary to initiate H-strand synthesis O H [ 48–51 ]. Transcripts initiated at LSP provide RNA 3′-ends from which POLγ can initiate DNA synthesis. In human mitochondria, there are multiple transitions points (RNA-to-DNA transitions) located downstream of LSP, clustering around two conserved sequence motifs, CSB3 and CSB2 ( Figure 1 , top panel) [ 52–54 ]. These conserved sequence elements are guanine-rich and during transcription, a G-quadruplex structure can form between nascent RNA and the nontemplate DNA strand at CSB2. In this manner, the nascent transcript is anchored to mtDNA, forming an R-loop structure [ 30 , 55 ]. The G-quadruplex structure also causes premature transcription termination at sites roughly corresponding to RNA to DNA transition sites mapped in the CSB2-region [ 56 ]. Based on these observations, it was hypothesized that sequence-dependent transcription termination may be responsible for primer formation at O H [ 54–56 ]. The transcription elongation factor TEFM, strongly reduces transcription termination and R-loop formation at CSB2, leading to the suggestion that active TEFM may influence the ratio between primer formation and full-length, productive transcription [ 57 , 58 ]. Arguing against this idea, knockdown of TEFM in cells only has very limited effects on mtDNA copy number and mitochondrial replication intermediates [ 59 ]. In addition, there are no direct experimental evidence demonstrating that R-loop-forming, prematurely terminated transcripts can be directly used by POLγ to initiate DNA synthesis. Further experiments are clearly needed to define the precise role of R-loops and TEFM in replication initiation.

The mechanisms of DNA replication initiation at O H may resemble those previously described for initiation of DNA replication in the E. coli plasmid ColE1. In the plasmid, a transcript denoted RNAII associates with the template strand, forming an R-loop that is used to prime DNA synthesis [ 60 , 61 ]. Furthermore, the ColE1 origin of replication is situated downstream of a guanine-rich stretch that is essential for both replication initiation and R-loop formation [ 61 , 62 ]. In ColE1, the R-loop is cleaved by RNase H, before it is used to prime DNA synthesis. If the mitochondrial RNASEH1 plays a similar role in mammalian cells remains to be determined [ 63 , 64 ].

Termination of mtDNA replication

When POLγ has completed synthesis, the newly formed DNA strands are ligated by DNA ligase III [ 65 , 66 ]. To allow for efficient ligation, the 5′- and 3′-ends of the nascent DNA ends must be juxtaposed, which means that the RNA primers used to initiate mtDNA synthesis must first be removed [ 67 ]. A likely candidate for primer removal is RNASEH1, inasmuch as RNA primers are retained in the mitochondrial origin regions in mouse embryonic fibroblasts lacking Rnaseh1 and there is a loss of mtDNA in Rnaseh1 knockout mice [ 35 , 68 ].

After completing a full circle-replication, POLγ encounters the 5′-end of the nascent full-length mtDNA strand it has just produced. At this point, POLγ initiates successive cycles of polymerization and 3′–5′ exonuclease degradation at the nick [ 67 , 69 ]. This process, idling, is required for proper ligation. POLγ lacking exonuclease activity is unable to idle and instead continues DNA synthesis into the dsDNA region past the 5′-end, thereby creating a flap-structure that cannot be ligated. Failure to create ligatable DNA ends may explain why mice with exonuclease-deficient POLγ display strand-specific nicks at O H [ 67 , 70 ].

Interestingly, there is a major 5′-end of nascent DNA located approximately 100 bp further downstream of the identified RNA-to-DNA transitions sites. Historically, this site (position 191 in human mtDNA) has been seen as a part of O H , but how this 5′-end is generated is not clear [ 25 ]. Although initially identified as a start site for mtDNA replication, the free 5′-end at position 191 may be generated in other ways. For instance, the nascent H-strand may undergo considerable 5′-end processing during primer removal, removing not only the RNA primer, but also ∼100 nt of downstream DNA [ 42 ]. In this way, the site for RNA-to-DNA transition would be separated from the site of nascent H-strand ligation at the end of replication. A possible candidate for this effect is the mitochondrial genome maintenance exonuclease 1 (MGME1), a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily [ 71 , 72 ]. In vitro analysis revealed that MGME1 cuts both ssDNA and DNA flap substrates. Human cells lacking active MGME1 display impaired ligation at O H and the formation of linear deleted mtDNA molecules spanning O H and O L [ 72 ]. In addition, there are increased levels of 7S DNA. The 5′-ends of these 7S DNA products are located further upstream (i.e. closer to CSB2) than what is observed in normal cells, suggesting that MGME1 is involved in processing the 5′-end of the nascent H-strand. Further studies of MGME1 and how it works together with RNASEH1 to process nascent replication products will be important.

Separation mtDNA

During DNA replication, the parental molecule remains intact, which poses a steric problem for the moving replication machinery. Topoisomerases belonging to the type 1 family can relieve torsional strain formed in this way, by allowing one of the strands to pass through the other. In mammalian mitochondria, TOP1MT a type IB enzyme can act as a DNA “swivel”, working together with the mitochondrial replisome [ 73 ]. Knockout of the Top1mt gene in mouse generates viable offspring that shows altered mtDNA supercoiling [ 74 , 75 ].

In other systems, replication of intact, circular DNA generates daughter molecules linked together as catenanes , i.e. mechanically interlocked, but not yet completely finished DNA circles. Therefore, replication of circular genomes requires decatenation to generate complete daughter molecules separation ( Figure 3 ). The existence of catenanes in mitochondria was reported already in 1967 by Vinograd and co-workers, who identified mtDNA molecules linked together by X-type brances, which they suggested were formed during completion of mtDNA replication [ 76 ]. Recently, it was demonstrated that these X-type structures are hemicatenanes, i.e. double-stranded DNA molecules linked together via a single-stranded linkage [ 77 ]. A mitochondrial isoform of Topoisomerase 3α (Top3α) is required to resolve the hemicatenane structure ( Figure 3 ), inasmuch as loss of Top3α causes decrease in mtDNA and the formation of large, catenated mtDNA networks. Patient mutations that decrease Top3α activity cause symptoms similar to those caused by mutations in other mitochondrial replication factors, including muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia [ 77 ]. Interestingly, the hemicatenanes holding these mtDNA networks together are located to the O H -region, suggesting that these structures are formed during the completion of mtDNA replication. Even if the exact mechanisms remain unclear, previous theoretical work that has postulated ways by which hemicatenanes can be formed during replication of circular DNA molecules [ 78 ]. Further work is required to understand how mtDNA replication is terminated and hemicatenanes are formed.

After mtDNA replication, the new daughter molecules are mechanically linked via a hemicatenane structure, which requires Top3α to be resolved.

Even if Top3α is required for separation of newly replicated mtDNA, additional proteins are probably also needed. There is a nuclear isoform of Top3α that functions together with three other proteins; the helicase BLM and the OB-fold proteins RMI1 and RMI2. Together, these proteins form the BTR complex, which acts to dissolve double Holliday junctions, and Top3α requires the other subunits to exert full topoisomerase activity [ 79 ]. However, since neither BLM, RMI1, or RMI2 have mitochondrial isoforms, other proteins may partner with Top3α in mitochondria to regulate and/or stimulate its activity [ 77 ].

Nucleoid replication

mtDNA is not a naked molecule, but packaged into large nucleoprotein complexes, nucleoids [ 80 ], which can be visualized by various fluorescent microscopy approaches [ 81 ]. Studies have revealed that nucleoids have an average size of ∼100 nm in diameter [ 82 , 83 ] and in most cases there is a single mtDNA molecule per nucleoid [ 82 ]. The major structural protein component of the nucleoid is TFAM, which is present at a ratio of 1 subunit per 16–17 bp of mtDNA. TFAM is a member of the high mobility group (HMG) box domain family and it binds DNA without sequence specificity [ 84 ]. TFAM is also an essential component of the mitochondrial transcription machinery [ 85 ]. During transcription initiation, the protein binds upstream of the transcription start site and induces a sharp bend into DNA [ 86–88 ]. Nucleoid-like particles can be reconstituted by simply mixing TFAM and mtDNA, implying that TFAM on its own can fully compact mtDNA [ 89–91 ]. TFAM has two DNA binding sites, and appears to compact mtDNA by cross-strand binding and loop formation. In addition, TFAM binds DNA in a cooperative manner, forming protein-patches on mtDNA [ 90–92 ].

That TFAM acts as an epigenetic regulator of mtDNA replication is a tantalizing possibility. Super-resolution microscopy has revealed different forms of nucleoids [ 91 ]. Perhaps, the more compact nucleoids represent a mtDNA storage form, whereas the larger forms are involved in active replication and/or transcription. Nucleoids involved in active DNA replication have been localized to contact points between the endoplasmic reticulum (ER) and mitochondria. At these sites, mitochondrial division takes place, leading to the idea that contacts between the endoplasmic reticulum and mitochondria can coordinate mtDNA synthesis with division to ensure even distribution of newly replicated nucleoids within the mitochondrial network [ 93 ].

In vitro , small changes in the TFAM to DNA ratio can have dramatic consequences. At physiological ratios, there are large variations in mtDNA compaction, fully compacted nucleoids and naked DNA can be observed simultaneously. Under these conditions, a small increase in TFAM concentrations can dramatically increase the number of full compacted mtDNA molecules. [ 89 , 90 ]. This model may explain why TFAM levels in vivo remain roughly proportional to mtDNA levels, and suggests that relatively small changes in TFAM concentrations can have strong effects on both gene expression and mtDNA replication. In support of this notion, longer patches of TFAM prevents DNA unwinding and as a consequence, the mtDNA replication and transcription machineries cannot progress [ 90 ]. TFAM may therefore function as an epigenetic regulator, which controls the number of mtDNA molecules available for active transcription and/or mtDNA replication.

Concluding remarks

A detailed understanding of mtDNA replication is not only important from a basic science point of view, but may also explain the formation of deletions and point mutations associated with human disease and aging. A detailed knowledge of these process may pave the way for development of new therapeutic strategies that can be of use in mitochondrial medicine.

• Mammalian mtDNA is replicated by proteins distinct from those used for nuclear DNA replication.
• According to the strand displacement model, replication is initiated from two distinct origins, OH and OL.
• Transcripts initiated at LSP provide the primer from which POLγ can initiate DNA synthesis at OH.
• O L forms a stem–loop structure and POLRMT initiates primer synthesis from the single-stranded loop region.
• The role of the mitochondrial D-loop is not understood.
• RNASEH1 and MGME1 play important roles in primer removal, but the details of this process are not fully understood.
• Top3α is required to resolve hemicatenane structures formed between new mtDNA molecules at the end of replication.
• mtDNA is not a naked molecule, but packaged into nucleoprotein complexes, nucleoids.
• Mitochondrial division is linked to active mtDNA synthesis.

Acknowledgments

I am grateful to Dr Jay P. Uhler who prepared the illustrations.

Abbreviations

Competing Interests

The author declares that there are no competing interests associated with the manuscript.

This work was supported by Swedish Research Council (2013-3621); Swedish Cancer Foundation (CAN2016/816); European Research Council (DELMIT); the IngaBritt and Arne Lundberg Foundation; and the Knut and Alice Wallenberg Foundation.

The Process of Semi-Conservative Replication ( AQA A Level Biology )

Revision note.

Biology Lead

The Process of Semi-Conservative Replication

• DNA replication occurs in preparation for mitosis , when a parent cell divides to produce two genetically identical daughter cells – as each daughter cell contains the same number of chromosomes as the parent cell, the number of DNA molecules in the parent cell must be doubled before mitosis takes place
• DNA replication occurs during the S phase of the cell cycle (which occurs during interphase , when a cell is not dividing )
• The enzyme helicase   unwinds  the DNA double helix by breaking the hydrogen bonds between the base pairs on the two antiparallel polynucleotide DNA strands to form two single polynucleotide DNA strands
• Each of these single polynucleotide DNA strands acts as a template for the formation of a new strand made from free nucleotides that are attracted to the exposed DNA bases by base pairing
• The new nucleotides are then joined together by DNA polymerase which catalyses condensation reactions to form a new strand
• The original strand and the new strand joined together through hydrogen bonding between base pairs to form the new DNA molecule
• This method of replicating DNA is known as semi-conservative replication because half of the original DNA molecule is kept ( conserved ) in each of the two new DNA molecules

Semi-conservative replication of DNA

DNA Polymerase

• These nucleotides are known as nucleoside triphosphates or ‘activated nucleotides’
• The extra phosphates activate the nucleotides, enabling them to take part in DNA replication
• The bases of the free nucleoside triphosphates align with their complementary bases on each of the template DNA strands
• The enzyme DNA polymerase synthesises new DNA strands from the two template strands
• It does this by catalysing condensation reactions between the deoxyribose sugar and phosphate groups of adjacent nucleotides within the new strands, creating the sugar-phosphate backbone of the new DNA strands
• DNA polymerase cleaves (breaks off) the two extra phosphates and uses the energy released to create the phosphodiester bonds (between adjacent nucleotides)
• Hydrogen bonds then form between the complementary base pairs of the template and new DNA strands

Nucleotides are bonded together by DNA polymerase to create the new complementary DNA strands

Leading & lagging strands

• DNA polymerase can only build the new strand in one direction (5’ to 3’ direction)
• As DNA is ‘unzipped’ from the 3’ towards the 5’ end, DNA polymerase will attach to the 3’ end of the original strand and move towards the replication fork (the point at which the DNA molecule is splitting into two template strands)
• This means the DNA polymerase enzyme can synthesise the leading strand continuously
• This template strand that the DNA polymerase attaches to is known as the leading strand
• The other template strand created during DNA replication is known as the lagging strand
• On this strand, DNA polymerase moves away from the replication fork (from the 5’ end to the 3’ end)
• This means the DNA polymerase enzyme can only synthesise the lagging DNA strand in short segments (called Okazaki fragments)
• A second enzyme known as DNA ligase is needed to join these lagging strand segments together to form a continuous complementary DNA strand
• DNA ligase does this by catalysing the formation of phosphodiester bonds between the segments to create a continuous sugar-phosphate backbone

The synthesis of the complementary strands occurs slightly differently on the leading and lagging template strands of the original DNA molecule that is being replicated

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Essay on DNA: Meaning, Features and Forms | Genetics

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In this article we will discuss about:- 1. Meaning of DNA 2. Features of DNA 3. Molecular Structure 4. Components 5. Forms.

• Essay on the Forms of DNA

Essay # 1. Meaning of DNA:

A nucleic acid that carries the genetic information in the cell and is capable of self-replication and RNA synthesis is referred to as DNA. In other words, DNA refers to the molecules inside cells that carry genetic information and pass it from one generation to the next. The scientific name for DNA is deoxyribonucleic acid.

Essay # 2. Features of DNA:

The main features of DNA are given below:

i. Location:

In eukaryotes, DNA is found both in nucleus and cytoplasm. In the nucleus it is a major component of chromosome, whereas in cytoplasm it is found in mitochondria and chloroplasts. In prokaryotes, it is found in the cytoplasm.

ii. Structure:

Mostly the DNA structure is double stranded in both eukaryotes and prokaryotes. However, in some viruses DNA is single stranded. DNA is a double stranded molecule held together by weak bonds between base pairs of nucleotides. The four nucleotides in DNA contain the bases: adenine (A), guanine (G), cytosine (C), and thymine (T).

iii. Shape:

In eukaryotes, the DNA is of linear shape. In prokaryotes and mitochondria; the DNA is circular.

iv. Replication:

The DNA is capable of self-replication. This is the only chemical which has self-replicating capacity. The DNA replicates in semi-conservative manner. In eukaryotes, DNA replicates during the S-Phase of the cell cycle.

There are different forms of DNA such as A, B, C, D and Z DNA. The DNA may be linear or circular. It may be double stranded or single stranded. It may be right handed or left handed. The DNA may be repetitive or unique. It may be nuclear or cytoplasmic.

vi. Functions:

DNA plays important role in various ways. It is used in transcription i.e. synthesis of mRNA which in turn is used in protein synthesis. It carries genetic information from one generation to the next generation. DNA stores and transmits the genetic information in cells.

It forms the basis for genetic code. The genes are made of DNA and are responsible for passing on traits from generation to generation. DNA contains the genetic instructions for the development and functioning of living organisms. Thus it is the substance of heredity.

Essay # 3. Molecular Structure of DNA :

The double helical structure of DNA was identified by Watson and Crick in 1953. This brilliant research work resulted in significant breakthrough in understanding the gene function. This structure has been verified in many different ways and is universally accepted. James Watson and Francis Crick were awarded Nobel prize in 1958 for this significant contribution in the field of molecular biology.

The important features of their model of DNA are as follows:

1. Two helical polynucleotide chains are coiled around a common axis, the chains run in opposite directions.

2. The purine and pyrimidine bases are on the inside of the helix whereas the phosphate and deoxyribose units are situated on the outside. Also the planes of the base residues are perpendicular to the helix axis. While the planes of the sugar residues are almost at right angles to those of the bases.

3. The diameter of the helix is 2 nm. Adjacent bases are separated by 0.34 nm along the helix axis. Hence the helix repeats itself every 10 residues on each chain at intervals of 3.4 nm.

4. The two chains are held together by hydrogen bonds formed between pairs of bases. Pairing is highly specific. Adenine pairs with thymine, guanine always pairs with cytosine. A = T, G = C.

5. The sequence of bases along the polynucleotide chain is not restricted. The precise sequence of bases carries the genetic information.

6. The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a spiral staircase.

7. The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase.

Essay # 4. Components of DNA :

DNA molecule is a polymer which is composed of several thousand pairs of nucleotide monomers. Union of several nucleotides together leads to the formation of polynucleotide chain. The monomer units of DNA are nucleotides, and the polymer is known as a “polynucleotide.” Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group.

There are three components of DNA, viz:

(1) Nitrogenous bases,

(2) Deoxyribose sugar, and

(3) Phosphate group.

These are briefly discussed below.

i. Nitrogenous Bases :

Nucleotides are also known as nitrogenous bases or DNA bases. Nitrogenous base are of two types, viz. pyrimidines and purines.

Pyrimidines:

Main features of pyrimidines are given below:

(i) These are single ring structures,

(ii) These are of two types namely cytosine and thymine,

(iii) They occupy less space in DNA structure,

(iv) Pyrimidine is linked with deoxyribose sugar at position 3.

Main features or purines are given below:

(i) They are double ring compounds.

(ii) They are of two types, viz. adenine and guanine.

(iii) They occupy more space in DNA structure.

(iv) Deoxyribose sugar is linked at position 9 of purine.

Thus, in DNA there are four different types of nitrogenous bases, viz. adenine (A), guanine (G), cytosine (C) and thymine (T). In RNA, the pyrimidine base thymine is replace by uracil.

Base Pairing:

The purine and pyrimidine bases always pair in a definite fashion. Adenine will always pair with thymine and guanine with cytosine. Adenine and thymine are joined by double hydrogen bonds while guanine and cytosine are joined by triple hydrogen bonds. However, these bonds are weak which help in separation of DNA strands during replication.

ii. Deoxyribose Sugar :

This is a pentose sugar having five carbon atoms. The four carbon atoms are inside the ring and the fifth one is with CH 2 group. This has three OH groups on 1, 3 and 5 carbon positions. Hydrogen atoms are attached to carbon atoms one to four. In RNA, the sugar ribose is similar to deoxyribose except that it has OH group on carbon atom 2 instead of H group.

iii. Phosphate :

The phosphate molecule is arranged in an alternate manner to deoxyribose molecule. Thus there is deoxyribose on both sides of phosphate. The phosphate is joined with carbon atom 3 of deoxyribose at one side and with carbon atom 5 of deoxyribose on the other side.

Nucleosides and Nucleotides :

A combination of deoxyribose sugar and nitrogenous base is known as nucleoside and a combination of nucleoside and phosphate is called nucleotide.

Nucleoside = Deoxyribose Sugar + Nitrogenous base

Nucleotide = Deoxyribose + Nitrogenous base + Phosphate

Thus, a nucleotide is a nucleoside with one or more phosphate groups covalently attached to it. Nucleosides differ from nucleotides in that they lack phosphate groups. The four different nucleosides of DNA are deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), and deoxythymidine (dT).

DNA Backbone :

The DNA backbone is a polymer with an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3′-hydroxyl and 5′-hydroxyl groups to phosphate groups in ester links, also known as “phosphodiester” bonds.

Essay # 5. Forms of DNA :

Depending upon the nucleotide base per turn of the helix, pitch of the helix, tilt of the base pair and humidity of the sample, the DNA can be observed in four different forms namely, A, B, C and D. The comparison of A, B and Z forms of DNA is presented in Table 15.1.

i. B-form :

This is the same form of DNA proposed by Watson and Crick.

Main features of B form of DNA are given below:

1. This is the most common form of DNA.

2. It is observed when humidity is 92% and salt concentration is high.

3. The coiling is in the right direction.

4. The number of base is 10 per turn of helix.

5. The pitch is 3.4 nm.

6. The sugar phosphate linkage is normal.

7. The helix is narrower and more elongated than A form.

8. The major groove is wide which is easily accessible to proteins.

9. The minor groove is narrow.

10. The conformation is favored at high water concentrations.

11. The base pairing is nearly perpendicular to helix axis.

12. The sugar puckering is C2′-endo.

ii. A-form :

1. This form is observed when the humidity of the sample is 75%.

2. The coiling is in the right direction.

3. The number of bases is 10.7 per turn of helix.

4. The pitch is 2.8 nm.

5. The sugar phosphate linkage is normal.

6. The major groove is deep and narrow which is not easily accessible to proteins.

7. The minor groove is wide and shallow which is accessible to proteins, but information content is lower than major groove.

8. The helix is shorter and wider than B form.

9. The conformation is favored at low water concentrations.

10. The base pairs are tilted to helix axis.

11. The sugar puckering is C3′-endo.

iii. Z-form :

1. The helix has left-handed coiling pattern.

2. The number of bases is 12 per turn of helix.

3. The pitch is 4.5 nm.

4. The sugar phosphate linkage is zigzag.

5. The major “groove” is not really a groove.

6. The minor groove is narrow.

7. The helix is narrower and more elongated than A or B form.

8. The base pairing is nearly perpendicular to helix axis.

9. The conformation is favored by high salt concentrations.

10. The sugar puckering is C: C2 endo, G : C2 exo.

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• Structural Features of DNA | Genetics
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Mitochondrial DNA replication in mammalian cells: overview of the pathway

Affiliation.

• 1 Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, 405 30 Gothenburg, Sweden [email protected].
• PMID: 29880722
• PMCID: PMC6056714
• DOI: 10.1042/EBC20170100

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome and a dedicated DNA replication machinery is required for its maintenance. Many disease-causing mutations affect mitochondrial replication factors and a detailed understanding of the replication process may help to explain the pathogenic mechanisms underlying a number of mitochondrial diseases. We here give a brief overview of DNA replication in mammalian mitochondria, describing our current understanding of this process and some unanswered questions remaining.

Keywords: DNA helicase; DNA polymerase; DNA replication; mitochondrion.

© 2018 The Author(s).

PubMed Disclaimer

Conflict of interest statement

The author declares that there are no competing interests associated with the manuscript.

Figure 1. Map of human mtDNA

The genome encodes for 13 mRNA (green), 22 tRNA…

Figure 2. Replication of the human mitochondrial…

Figure 2. Replication of the human mitochondrial genome

Mitochondrial DNA replication is initiated at O…

Figure 3. Separation and segregation of the…

Figure 3. Separation and segregation of the human mitochondrial genome

After mtDNA replication, the new…

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The function of h2a histone variants and their roles in diseases.

1. Introduction

2. the functions of h2a histone variants, 2.1. gene transcription, 2.2. dna damage repair, 3. the roles of histone h2a variants in diseases, 3.1. cancer.

3.2. Embryonic Development Abnormalities

3.3. other diseases, 4. conclusions.

Author Contributions

Data availability statement, conflicts of interest.

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Yin, X.; Zeng, D.; Liao, Y.; Tang, C.; Li, Y. The Function of H2A Histone Variants and Their Roles in Diseases. Biomolecules 2024 , 14 , 993. https://doi.org/10.3390/biom14080993

Yin X, Zeng D, Liao Y, Tang C, Li Y. The Function of H2A Histone Variants and Their Roles in Diseases. Biomolecules . 2024; 14(8):993. https://doi.org/10.3390/biom14080993

Yin, Xuemin, Dong Zeng, Yingjun Liao, Chengyuan Tang, and Ying Li. 2024. "The Function of H2A Histone Variants and Their Roles in Diseases" Biomolecules 14, no. 8: 993. https://doi.org/10.3390/biom14080993

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