Structure-function analyses of Msh2-Msh6 and Msh2-Msh3 complexes: An insight into molecular mechanisms
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Mismatch repair (MMR) is a DNA repair pathway that repairs errors that occur spontaneously during DNA replication. It is thus an important pathway to maintain genome stability. In Saccharomyces cerevisiae, MutS homolouges (Msh) Msh2-Msh6 or Msh2-Msh3 initiate MMR. Msh2-Msh6 initiates repair of base-base mismatches and Msh2-Msh3 initiates repair of insertion/deletion loops (IDLs). Both of these complexes have an overlapping specificity for small IDLs. In addition to MMR, both Msh2-Msh6 and Msh2-Msh3 are involved in other DNA repair pathways. Msh2-Msh6 is involved in sensing extensive DNA methylation damage and signaling cell death in a pathway called DNA damage response (DDR). Msh2-Msh3 is involved in specialized forms of double strand break repair and is required in a step called 3' non-homologous tail removal (3'NHTR). Both Msh2-Msh6 and Msh2-Msh3 are involved in preventing recombination of homeologous sequences in a pathway called heteroduplex rejection. In this work we have examined the structural requirements of Msh2-Msh6 and Msh2-Msh3 in these different pathways. We first examined the role for Msh2 domain I for Msh2-Msh6 in MMR. We found that multiple factors insulate cells from defects in this part of Msh2 and specifically found that the N-terminal region (NTR) of Msh6 plays a compensatory role. We next examined the roles of these regions in Msh2-Msh6 function in DDR. We found that domain I of Msh2 compensates for the defects in the NTR of Msh6 in DDR but the NTR of Msh6 does not compensate for the lack of domain I of Msh2. We predict that while domain I of Msh2 is essential for complete functioning of Msh2-Msh6 in MMR and DDR, the NTR of Msh6 is more important for the complex's role in DDR. We next examined the requirements of Msh3 from it aromatic residues in its nucleotide binding pocket for Msh2-Msh3 function in MMR, 3'NHTR and heteroduplex rejection. We found that proper regulation of communication between the DNA binding and ATPase domains is essential for all three pathways. But we found that 3'NHTR and heteroduplex rejection were a lot less stringent in their requirements for the proper regulation of nucleotide binding by Msh3 when compared to MMR. We next performed an in vitro analysis of Msh2-Msh3 ATPase acitivity in the presence of substrates that resemble the DNA intermediates that Msh2-Msh3 binds to in the above pathways. We found that Msh2-Msh3 differentiates between the different substrates and the mechanisms of hydrolysis differ between the substrates. This is in support of the model that Msh2-Msh3 uses ATP to license repair.