DYNAMICS OF THE MISMATCH REPAIR COMPLEXES DURING DNA REPLICATION

Abstract

DNA mismatch repair (MMR) functions mainly to correct mispaired bases that escape the proofreading activity of the DNA polymerase during replication. Defects in MMR genes have been linked to compromised genome stability and diseases including cancer. MMR is a highly conserved process and the yeast Saccharomyces cerevisiae is an ideal model organism to explore aspects of MMR because of the ease of manipulation and homology to the human system. MMR initiates when a mismatch in the DNA helix is recognized by MutS homologs. Subsequent events include excision of the error-containing strand followed by re-synthesis. A critical step in this process is directing repair to the newly synthesized strand, which requires a strand discrimination signal. Current data suggest that transient discontinuities in the DNA backbone, known as nicks, generated during replication serve as the strand discrimination signal. Additionally, histones have the capacity to block mismatch recognition and are known to rapidly assemble behind the replication fork. Thus, there must be a short window of opportunity for the MutS homologs to scan for mismatches and access the strand discrimination signals during replication. To address these unresolved issues, we hypothesize that the MMR machinery tracks with the replisome to allow for efficient scanning and access to the strand discrimination signal. We employed chromatin immunoprecipitation and DNA tiling microarrays (ChIP-chip) to determine the distribution of the eukaryotic MutS complexes during replication. The data indicate that during S-phase of the cell cycle MutS binds origins of replication and shows bi-directional occupancy of regions flanking the origins over time with timing consistent with fork progression. Importantly, MutS displays the same origin binding and spreading pattern as the leading strand DNA polymerase over multiple experiments. In sum, our data supports the hypothesis of the MMR machinery tracking with the replisome.

There are two MutS complexes that occur in eukaryotes, MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3). Both complexes recognize the different types of mismatches that arise during DNA replication. Reporter constructs have traditionally been utilized to assay the types of mismatches targeted by each complex. With the availability of new techniques, we can now analyze the functions of MutSα and MutSβ on a genome wide scale. In this work, we used next generation sequencing to determine the mutation spectra in strains lacking MSH2, MSH3 or MSH6. Our studies confirm the findings of previous genetic experiments that MutSα and MutSβ are functionally redundant for repair at HPRs; however, each complex is essential for ~6-7% of the mismatches generated during replication. In this work, we provide evidence that both complexes should be in the vicinity of the replisome to ensure that majority of the mutations can be avoided during replication.