In this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC... Show moreIn this thesis the role of DNA mismatch repair (MMR) in the cellular response to several genotoxic agents is described. We show that MMR plays an important role in the protection against UVC-induced mutagenesis in mouse embryonic stem (ES) cells. UVC was shown to induce six times more mutations in mouse ES cells deficient for the mismatch recognition dimer MutSalpha compared to wild type cells. The Hprt mutational spectrum of UVC-induced mutations was similar in MutSalpha-proficient and MutSalpha-deficient mouse ES cells. We subsequently tried to gain insight into the mechanism by which MMR mediates protection against UVC-induced mutagenesis. We found that UVC induces a late S/G2-phase arrest which partially depends on the presence of MutSalpha. The MutSalpha-dependent late S/G2-phase arrest coincided with the appearance of phosphorylated Chk-1 of which the levels were higher in MutSalpha-proficient cells compared to MutSalpha-deficient cells. Importantly, abolishment of the UVC-induced late S/G2-phase arrest in both MutSalpha-proficient and MutSalpha-deficient cells did not result in a smaller difference in mutation induction between both genotypes after UVC treatment. We propose that MMR removes mismatches from UVC-induced compound lesions and that the MutSalpha-induced late S/G2-phase arrest is the result of the appearance of DNA single stranded regions arising during this process of MMR. Show less
The work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in... Show moreThe work described in this thesis deals with characterization of DNA binding by the BRCT domain of the large subunit of RFC. Replication Factor C (RFC) is a five protein complex involved in initiating and regulating new DNA synthesis. The first half of the thesis describes region of the RFC and structural determinants of DNA required for productive protein-DNA interaction. The second half describes three-dimensional structure determination of the protein-DNA complex, which consists of the BRCT region of the RFC and doubled stranded DNA. The resulting structure based on the data from NMR and mutagenesis reveals structural conservations of few amino acids among the members of BRCT domain superfamily, which are known to bind either phosphorylated peptide or DNA. The work may help us to identify other potential DNA binding BRCT domains. Show less
