The role of DNA replication in mismatch repair
Masih, Prerna Jasmine
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DNA Mismatch Repair (MMR) plays an important role in maintaining genomic fidelity and stability in all living organisms, ranging from bacteria to humans, and is known to be closely associated with DNA replication and present at DNA replication foci. MMR rapidly repairs mismatched base pairs preferentially in newly replicated DNA and on the newly synthesized daughter strand. MMR could be targeted to replicating DNA merely by the presence of the newly synthesized mismatches; however, it is not known whether other mechanisms may exist to preferentially target the MMR machinery to replicating DNA. One known DNA replication protein, proliferating cell nuclear antigen (PCNA), has been shown to interact with the MMR proteins, MSH3, MSH6 and MLH1. While interactions with PCNA have been proposed as a possible mechanism for targeting MMR to replicating DNA, PCNA is involved in the downstream excision and DNA resynthesis steps of MMR and these interactions may reflect PCNA's role in these latter stages of repair. Currently no direct evidence exists to support the hypothesis that interactions with PCNA are involved in preferentially targeting MMR to the replicating DNA. Here the in vitro Siman Virus 40 (SV40) DNA replication system was used to evaluate the association of MMR proteins with replicating DNA. We isolated replicating SV40 DNA/protein complexes using gel filtration and showed them capable of subsequently carrying out DNA synthesis. Known DNA replication factors, both cellular as well as the viral factor, large T antigen, were shown to be recruited to these isolated replicating DNA complexes in a replication origin-dependent manner. We also showed that the MMR proteins, MLH1, MSH3 and MSH2, were also specifically recruited to the replicating DNA complexes. The role of polynucleotide synthesis in MMR recruitment was also evaluated, and polynucleotide synthesis was shown to be necessary. The PCNA inhibitor, p21, also inhibited the recruitment of PCNA as well as MutS complexes (MSH2 was used a marker protein for MutS complexes as it is a common subunit of MutS complexes) to SV40 replicating DNA/protein complexes. Furthermore, once DNA synthesis was allowed to progress, addition of p21 left PCNA associated with the replication intermediates, but resulted in dramatic decrease in association of MutS complexes. Together, these results suggest that the vast majority of the MutS complexes associated with replicating DNA is PCNA-dependent and moreover dependent on the availability of multi-protein binding sites on PCNA.