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How exactly does DNA polymerase III detect a mismatched base?


How exactly does DNA polymerase III detect a mismatched base? I know how it removes it, via exonuclease activity, but how does it 'detect' it molecularly in the first place?


How exactly does DNA polymerase III detect a mismatched base? - Biology

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 1). 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.

Figure 1. Proofreading by DNA polymerase corrects errors during replication.

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 2). 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.

Figure 2. In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

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 3). 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.

Figure 3. Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.


How exactly does DNA polymerase III detect a mismatched base? - Biology

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 just been added (Figure 1). 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.

Figure 1. Proofreading by DNA polymerase corrects errors during replication.

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 2). 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.

Figure 2. In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

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 3).

Figure 3. Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

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.


DNA Repair

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 1). 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 (Figure 1).

Figure 1 Proofreading by DNA polymerase corrects errors during replication. Photo credit Madeline Price Ball Wikimedia.

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 2). 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 (CH3) 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.

Figure 2 In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

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 3). 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 thymine-thymine dimers (the small – connecting the two Ts in Figure 3).

Figure 3 Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa (Figure 4). 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 have a higher risk of contracting skin cancer than those who don’t have the condition.

Figure 4 Xeroderma pigmentosa is a condition in which thymine dimerization from exposure to UV is not repaired. Exposure to sunlight results in skin lesions. (credit: James Halpern et al.)

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 a mutagen: 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. Other mutations can have serious effects on the organism (such as the mutation that causes xeroderma pigmentosa.

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 gametes. 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 a gamete, the mutation can be passed on to the next generation.


MMR mediates DNA damage signaling

MMR deficiency and drug resistance

DNA-damaging agents such as the alkylating agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), temozolomide, or procarbazine are cytotoxic agents that kill most of the replicating cells. Many cancer therapeutics are genotoxic and cytotoxic agents that induce apoptotic cell death. Interestingly, many cells that acquire resistance to such agents are deficient in MMR. For example, the human lymphoblastoid cell line MTI, which has a defect in hMSH6, was derived by culturing TK6 cells in the presence of a high concentration of MNNG. The resulting MNNG-resistant MT1 cells are defective in strand-specific MMR 76 . Many human colorectal cancer cell lines are also resistant to alkylating agents and have associated defects in MMR. The causal relationship between drug resistance and MMR is demonstrated by the fact that hMLH1-defective MNNG-resistant cells lose drug resistance when the hMLH1 defect is genetically complemented with wild-type hMLH1 on chromosome 3 77 . It has also been observed that defects in MSH2 and PMS2 confer resistance to alkylating agents (reviewed in 78 ). The mechanism by which MMR influences drug cytotoxicity is discussed further below.

Resistance to methotrexate (MTX) has also been associated with phenotypic changes in MMR in human cells. This occurs by the unusual mechanism of co-amplification of the human chromosomal region that encodes dihydrofolate reductase (DHFR, the target of MTX) and hMSH3 79, 80 . Amplification of DHFR lowers sensitivity to MTX by overexpressing the target of the drug. However, overexpression of hMSH3 sequesters hMSH2 in the hMutSβ heterodimer, effectively preventing formation of the hMutSα (hMSH2/hMSH6) heterodimer, which leads to degradation of uncomplexed hMSH6, significant dysregulation of MMR and hypermutability 81, 82 . Overexpression of DHFR combined with genome-wide hypermutability and defective MMR are likely responsible for the MTX resistance of HL60 and other tumor cells.

MMR proteins promote DNA damage-induced cell cycle arrest and apoptosis

Cell cycle arrest is an important mechanism for preventing DNA damage-induced genomic instability. A large number of studies have characterized the so-called G2 or S phase checkpoints, and identified proteins required for cell cycle arrest, including ATM, ATR, p53, p73, Chk1, and Chk2. However, it was a somewhat unexpected finding that hMutSα- and hMutLα-deficient cells are defective in cell cycle arrest in response to multiple types of DNA damaging agents 6, 7, 83 . While the molecular basis of this effect is not precisely known, it has been reported that MMR-deficient cells fail to phosphorylate p53 and p73 in response to DNA damage 84, 85 . This implicates ATM, ATR, and/or c-Abl, because these kinases phosphorylate p53 and p73 during the response to DNA damage 85, 86 . In support of this, it has been reported that hMutSα and hMutLα interact physically with ATM, ATR-ARTIP, c-Abl, and p73 in cells treated with DNA damaging agents/drugs 83, 87, 88, 89 . These observations implicate hMutSα and hMutLα in a signaling cascade that leads from DNA damage to cell cycle arrest and/or apoptosis. They also at least in part explain the fact that drug-induced cytotoxicity is lost in MMR-deficient cells, as discussed above 6 . Very recently, EXO1 has been shown to be essential for upstream induction of DNA damage response, possibly by reducing ssDNA formation and recruiting RPA and ATR to the damage site 90 . It remains to be seen if MutSα and/or MutLα act to recruit EXO1 in DNA damage response as they do in MMR.

Two models have been proposed to describe the role of MMR in DNA damage signaling. The “futile DNA repair cycle” model (Figure 2, left) proposes that strand-specific MMR, which targets only newly replicated DNA, engages in a futile DNA repair cycle when it encounters DNA lesions in the template strand, and this futile cycling activates DNA damage signaling pathways to induce cell cycle arrest and apoptosis 6 . Support for this model came from both in vivo and in vitro experiments. Stojic et al. 86 showed that exposure to MNNG induces DNA breaks/gaps, cell cycle arrest, and persistent nuclear foci at sites of DNA damage. The DNA damage-associated repair foci contain both damage signaling and DNA repair proteins, including ATR, γ-H2AX, and RPA. York and Modrich 91 showed that nicked circular heteroduplex plasmid DNA containing a single O 6 -methylguanine (O 6 -me-G)-thymine (T) mispair cannot be repaired by the MMR system when the lesion (O 6 -me-G) and the nick are on opposite strands this suggests a futile repair process. An alternative model, referred to as the direct signaling model (Figure 2, right), argues that hMutSα/hMutLα directly trigger DNA damage signaling by recruiting ATM or ATR/ARTIP to the lesion, which activates a checkpoint response. This model is supported by an elegant study from the Hsieh laboratory showing that ATR and ATRIP form a complex with MutSα/MutLα in the presence of O 6 -me-G/T, which activates the ATR kinase and phosphorylates Chk1 89 . Because mammalian MMR proteins interact with a broad spectrum of DNA lesions 6 , this model is consistent with the notion that MutSα/MutLα acts as a sensor for DNA damage in mammalian cells. Both models provide a reasonable explanation for decreased DNA damage-induced apoptotic signaling and increased drug resistance in MMR-deficient cells.

Models for MMR-dependent DNA damage signaling. The “futile DNA repair cycle” model (left) suggests that DNA adducts (solid black circle) induce misincorporation, which triggers the strand-specific MMR reaction. Since MMR only targets the newly synthesized strand for repair, the offending adduct in the template strand cannot be removed, and will provoke a new cycle of MMR upon repair resynthesis. Such a futile repair cycle persists and activates the ATR and/or ATM damage signaling network to promote cell cycle arrest and/or programmed cell death. The direct signaling model proposes that recognition of DNA adducts by MSH-MLH complexes allows the proteins to recruit ATR and/or ATM to the site, activating the downstream damage signaling.


How exactly does DNA polymerase III detect a mismatched base? - Biology

Most mistakes during replication are corrected by DNA polymerase during replication or by post-replication repair mechanisms.

Learning Objectives

Explain how errors during replication are repaired

Key Takeaways

Key Points

  • Mismatch repair enzymes recognize mis-incorporated bases, remove them from DNA, and replace them with the correct bases.
  • In nucleotide excision repair, enzymes remove incorrect bases with a few surrounding bases, which are replaced with the correct bases with the help of a DNA polymerase and the template DNA.
  • When replication mistakes are not corrected, they may result in mutations, which sometimes can have serious consequences.
  • Point mutations, one base substituted for another, can be silent (no effect) or may have effects ranging from mild to severe.
  • Mutations may also involve insertions (addition of a base), deletion (loss of a base), or translocation (movement of a DNA section to a new location on the same or another chromosome ).

Key Terms

  • mismatch repair: a system for recognizing and repairing some forms of DNA damage and erroneous insertion, deletion, or mis-incorporation of bases that can arise during DNA replication and recombination
  • nucleotide excision repair: a DNA repair mechanism that corrects damage done by UV radiation, including thymine dimers and 6,4 photoproducts that cause bulky distortions in the DNA

Errors during Replication

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

Mutations: In this interactive, you can “edit” a DNA strand and cause a mutation. Take a look at the effects!

Most of the mistakes during DNA replication are promptly corrected by DNA polymerase which proofreads the base that has just been added. 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 correct 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 incorrect 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.

DNA polymerase proofreading: Proofreading by DNA polymerase corrects errors during replication.

Some errors are not corrected during replication, but are instead corrected after replication is completed this type of repair is known as mismatch repair. 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 incorrectly-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 been completed.

Mismatch Repair: In mismatch repair, the incorrectly-added base is detected after replication. The mismatch-repair proteins detect this base and remove it from the newly-synthesized strand by nuclease action. The gap is now filled with the correctly-paired base.

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. 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.

DNA Ligase I Repairing Chromosomal Damage: DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. A special enzyme, DNA ligase (shown here in color), encircles the double helix to repair a broken strand of DNA. DNA ligase is responsible for repairing the millions of DNA breaks generated during the normal course of a cell’s life. Without molecules that can mend such breaks, cells can malfunction, die, or become cancerous. DNA ligases catalyse the crucial step of joining breaks in duplex DNA during DNA repair, replication and recombination, and require either Adenosine triphosphate (ATP) or Nicotinamide adenine dinucleotide (NAD+) as a cofactor.

Nucleotide Excision Repairs: Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

DNA Damage and Mutations

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: 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, known as a deletion. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome.


DNA Replication Across Taxa

J.S. Lewis , . N.E. Dixon , in The Enzymes , 2016

Abstract

DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC , contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β 2 sliding clamp that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit clamp loader complex that loads β2 onto primer–template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein–protein and protein–DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication.


VISUAL CONNECTION

Figure 3: A replication fork is formed by the opening of the origin of replication, and helicase separates the DNA strands. An RNA primer is synthesized, and is elongated by the DNA polymerase. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches. The DNA fragments are joined by DNA ligase (not shown).

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 answer is ligase, as this enzyme joins together Okazaki fragments.

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 4) 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.

Figure 4: The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

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 5) received the Nobel Prize for Medicine and Physiology in 2009.

Figure 5: Elizabeth Blackburn, 2009 Nobel Laureate, was the scientist who discovered how telomerase works. (credit: U.S. Embassy, Stockholm, Sweden)

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 1 summarizes the differences between prokaryotic and eukaryotic replications.

Table 1: Differences between Prokaryotic and Eukaryotic Replications
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
Chromosome structure circular linear
Telomerase Not present Present


Mismatch Repair Pathway in Prokaryotes:

  1. In E-Coli mismatches are detected by a dimer of the mismatch repair protein MutS. MutS scan the DNA, recognizing the mismatches from the distortion they cause in the DNA backbone.
  2. MutS embraces the the mismatch-containing DNA , inducing a kink in the DNA and a conformational change in MutS itself.
  3. This complex of MutS and mismatch containing DNA recruits MutL, a second component of mismatch repair system .
  4. MutL, in turn, activates the mutH, an enzyme that causes incision or nick on one strand near the site of the mismatch.
  5. Nicking is followed by the action of specific helicase and one of three exonucleases. The helicase unwind the DNA, starting from the incision and moving in the direction of the site of the mismatch, and exonucleases progressively digest the displaced single strand, extending to and beyond the site of the mismatched nucleotide.
  6. This action produces a single-stranded gap, which is then filled by DNA polymerase III and sealed with DNA Ligase.

  • How does the mismatch repair system know which of the two mismatched nucleotides to replace?

The E-Coli enzyme Dam methylase methylates A residue on both strands of the sequence 5′-GATC-3′. The GATC sequence is widely distributed along entire genome , and all of these sites are methylated by the Dam methylase. When replication fork passes through DNA that is methylated at GATC sites on both strands , the resulting daughter DNA duplex will have hemimethylated (that is methylated on only parent strand). Thus for few minutes, until the Dam mathylase catches up and methylates the newly synthesized strand, daughter DNA duplex will be methylated only on the strand that served as the template. Thus the newly synthesized strand lacks methyl group and are so marked and hence can be recognized as the strand for repair.

The MutH proteins binds at such hemimethylated sites, but its endonuclease activity is normally latent. Only when it is contacted by MutL and LutS located at a nearby mismatch does MutH become activated. Once activated, MutH selectively nicks the unmethylated strand.


  1. This process fixes mistakes made during DNA replication. During replication in S phase, DNA polymerase can sometimes add an incorrect nucleotide as it duplicates the DNA.
  2. DNA polymerase can actually detect this error and remove the incorrect nucleotide. This is called DNA polymerase proofreading
  3. DNA polymerase proofreading occurs when DNA polymerase uses its exonuclease active site to remove the most recently added nucleotide and then replaces it with the correct nucleotide.
  4. Note that DNA polymerase can only correct the previously added nucleotide, but not any nucleotides prior to that.

Mismatch repair

  1. If an error is not corrected by DNA polymerase proofreading it can still be caught by mismatch repair.
  2. Mismatch repair begins when an enzyme complex scans the DNA. One component of the complex recognizes distortion caused by mismatched base pairing (for example if A was incorrectly paired with C in the duplicated DNA).
  3. Another component of the enzyme complex acts like a pair of scissors and snips the DNA near the mistake and removes the area surrounding the mistake.
  4. Next, a DNA polymerase returns to fill in the now empty area with new nucleotides.
  5. Lastly, DNA ligase repairs the gap in the DNA backbone.

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Watch the video: La duplicazione del DNA HD (December 2021).