Mismatch Repair System

The replication machinery achieves remarkably high degree of accuracy using a proofreading mechanism. Proofreading improves the fidelity of DNA replication by a factor about 100 . The proof reading exonuclease is not, however, foolproof. Some mis incorporated nucleotides escape detection and become a mismatch between the newly synthesized strand and the template strand.

If the misincoporated nucleotide in the first round of replication is not subsequently detected and replaced, the sequence change will become permanent in the genome in the second round of replication.  At this point the mismatch will no longer exist; instead it will have resulted in a permanent change in the DNA sequence.

Fortunately, a mechanism exists for detecting the mismatches and repairing them, known as Mismatch Repair System. This system increases the accuracy of DNA synthesis by an additional two to three orders of magnitude.

Aims of Mismatch repair system are as follows:

  • Quick response : It must scan the genome for mismatches. Because mismatches are transient (they are eliminated in the second of replication) the mismatch repair system must find and repair mismatches.
  • Accuracy: The system must correct the mismatch accurately. that is it must replace the misincoporated nucleotides in the newly synthesized strand and not the correct nuceotide in the parent strand.

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.

Mismatch repair system in Eukaryotes:

Eukaryotes cells also repair mismatches and do so using homologs to MutS (Called MSH proteins for MutS homologs) and MutL (called MLH and PMS). Indeed, eukaryotes have multiple MutS-like proteins with different specificities.

Even though eukaryotic cells have MMR, mismatch repair systems, they lack MutH and E-coli’s clever trick of using hemimethylation to tag the parent strand.

Following are the detailed view how Eukaryotic Mismatch repair system works.

All eukaryotic organisms, including yeast, mouse and human, have MutS homologs (MSHs) and MutL homologs (MLHs). The eukaryotic MMR system has been well conserved during the evolutionary process. However, in contrast to MutS and MutL in bacteria, which function as homodimers, in eukaryotes MSHs and MLHs form heterodimers with multiple proteins. Five highly conserved MSHs (MSH2 to MSH6) are present in both yeast and mammals.

MSH1, which is present in mitochondria, exists only in yeast . MSH4 and MSH5 show reproductive tissue- specific expression, and null mutations of these genes do not confer mutator phenotypes because they are involved in meiotic recombination but not postreplication repair. Genetic and biochemical studies have indicated that MSH2 is required for all mismatch correction in nuclear DNA, whereas MSH3 and MSH6 are required for the repair of some distinct and overlapping types of mismatched DNA during replication. These three MutS homologs make two heterodimers: MutSa (MSH2⁄MSH6) and MutSb (MSH2⁄ MSH3). The former plays the major role in recognition of mismatched DNA in eukaryotic MMR. That is, MutSa functions in the repair of base–base mispairs as well as a range of insertion ⁄ deletion loop mispairs, whereas MutSb primarily functions in the repair of insertion ⁄ deletion loop mispairs.

MutL homologs in eukaryotic organisms were identified as genes whose amino acid sequences showed high similarity with prokaryotic MutL proteins, or whose mutation phenotypes were increased levels of postmeiotic segregation (PMS) that resulted from a failure to repair mismatches in meiotic recombination intermediates. There are four homologs of MutL in both yeast and mammals. In a genetic analysis, defects in MLH1 and PMS1 in yeast resulted in more severe mutator phenotypes, reminiscent of those of MSH2 and MSH6, than defects in the two other MutL homologs. Also, MLH1 interacted with the other three MutL homologs in a yeast two-hybrid analysis. Overall, yeast MLH1⁄PMS1 and mammalian MLH1⁄PMS2 heterodimers (each known as MutLa) play a major role in mutation avoidance, and the other two heterodimers of MutL homologs take part in the repair of specific classes of mismatches. The biochemical activities and structure of MutL homologs are closely related to those of prokaryotic MutL proteins, especially in the N-terminal domain. The X-ray crystallographic structure of the conserved N-terminal 40-kDa fragment of human PMS2 resembles that of the ATPase fragment of E. coli MutL.

Extensive genetic studies in yeast have failed to find orthologs of MutH and UvrD in the MMR system, and there may be no homolog of these two proteins in the eukaryotic genome. Therefore, some divergence in the MMR system from strand discrimination and the nicking process might occur between prokaryotes and eukaryotes.

A recent increase in our knowledge of the eukaryotic MMR system provides some understanding of this divergence. In mammalian cell extracts, mismatches provoke initiation of excision at pre-existing nicks in exogenous DNA substrates with high efficiency and specificity. The molecular nature of eukaryotic MMR could be assessed using cell extract assays in vitro, and components of the eukaryotic MMR system have been identified with depletion and complementation assays using cell extracts. One protein, identified in this way, is proliferating cell nuclear antigen (PCNA). PCNA is known to function as a processivity factor for replicative polymerase, but some mutations in the PCNA gene result in mutator phenotypes, and its interactions with MSH2 and MLH1, and with MSH6, suggest that it functions in MMR. PCNA has biochemical activity that increases the binding of MutSa to mismatched DNA; the interactions between PCNA and MSH6 are essential for this biochemical activity, which suggests that PCNA might play a role in MMR at the mispair recognition stage. PCNAhas been proposed to function in the mismatch recognition stage of MMR by helping MutSa search for mismatched DNA or increasing the mismatchbinding specificity of MutSa. One intriguing point about the role of PCNA in eukaryotic MMR is that the requirement for PCNA depends on the direction of the nick in the in vitro MMR assay. Although PCNA is required for mismatch-provoked excision directed by a 3’ strand break in HeLa nuclear extracts, it is not essential for excision directed by a 5’ nick. Moreover, whereas 3’ nick-directed excision iscompletely abolished by the inhibition of PCNA, 5’ nick-directed excision is affected only minimally.

Finally, a mismatch-provoked 5’ to 3’ excision reactioncan be reconstituted in a purified system that comprises only MutSa, MutLa, ExoI and replication protein A (RPA), without PCNA, and the process is similar to that observed in nuclear extracts. RPA, the eukaryotic single-stranded DNA-binding protein, has been shown to enhance excision and stabilize excision intermediates in crude fractions. The activities of ExoI are described below.

Genetic studies in yeast, and biochemical studies of MMR activity in cell extracts, indicate that eukaryotes use a mechanism similar to prokaryotes, with both 3’ to 5’ and 5’ to 3’ exonuclease activities for mismatch correction. ExoI, a 5’ to 3’ exonuclease, was found to play a role in mutation avoidance and mismatch repair in yeast, and its physical interaction with MSH2 and MLH1 also support a role in MMR. Intriguingly, the mammalian ExoI was reported to be involved in both 5’- and 3’ nick-directed excision in extracts of mammalian cells, but how ExoI can have a 3’ to 5’ exonuclease activity was unclear. Recent research by the Modrich group provides a plausible answer to this question [49]. They reconstituted mismatch-provoked excision, directed by a strand break located either 3’ or 5’ to the mispair, in a defined human system using purified human proteins. In the presence of the eukaryotic clamp loader replication factor C (RFC) and PCNA, 3’ to 5’ excision was supported by MutSa, MutLa, ExoI and RPA. Moreover, RFC and PCNA act to suppress 5’ to 5’ excision when the strand break that directs hydrolysis is located 3’ to the mismatch, which suggests that the polarity of mismatch-provoked excision by ExoI is regulated by PCNA and RFC. Once the strand is excised beyond the mismatch, DNA resynthesis occurs by the activity of polymerase d in the presence of PCNA and RPA. The remaining nick is then sealed by an as-yet-unidentified ligase, completing the repair process.

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