Analysis Of The Movement Of Saccharomyces Cerevisiae Mismatch Repair Proteins On Dna
Replication errors that escape DNA polymerase proof-reading activity are efficientl y recognized and repaired by conserved DNA mismatch repair factors. The overall result is a drastic reduction in deletion mutations. The mechanistic details of how mismatch repair proteins execute mismatch removal have not been elucidated. The aim of my thesis is to better understand how mismatch repair factors interact with DNA in order to identify mismatch sites. My work reveals that the mismatch repair complex, MLH1-PMS1, has unique DNA diffusion characteristics facilitated by structural features of the two subunits. Through bulk assays and total internal reflectance fluorescence microscopy (TIRFM), I found that MLH1PMS1 could independently bind DNA and rapidly diffuse using the thermal energy of the system. Furthermore, MLH1-PMS1 was shown to be the first passively diffusing protein that could bypass stationary nucleosomes. In contrast, the DNA diffusion activity of the mismatch recognition complex MSH2-MSH6 was blocked by nucleosomes. The timing and nature of mismatch repair is linked with replication and is thus proposed that the differences seen for the two complexes have important implications for repair in the context of the chromatin state directly at the replication fork. Each subunit of the MLH1-PMS1 complex is composed of two defined globular domains connected by an unstructured linker arm. The linker arms of the complex are proposed to facilitate topological DNA binding and diffusion along DNA in a hopping/stepping mechanism. I found that TEV protease cleavage within the linker arms of MLH1-PMS1 disrupted DNA binding and mismatch repair in vitro and in vivo. Using a genetic mismatch repair assay I found that shortening of the linker arms in MLH1 had a drastic effect on function whereas similar changes in PMS1 had little or no effect. Purified truncated complexes were able to interact with DNA and form ternary complexes with MSH2-MSH6 at a mismatch. Future studies should focus on the diffusion characteristic for these complexes. Together, my work has important implications for understanding how mismatch repair proteins can rapidly identify their targets in a chromatin landscape.
DNA mismatch repair; Replication; Cancer
Alani, Eric E
Collins, Ruth N.
Weiss, Robert S.
Ph. D., Biochemistry
Doctor of Philosophy
dissertation or thesis