Defects in DNA repair can lead to genome instability, a hallmark of cancer. The overall goal of this project is to study the molecular mechanisms of DNA repair in two model organisms: Escherichia coli and fission yeast Schizosaccharomyces pombe. Oxidative damage is a major source of mutation load in living organisms and plays a role in carcinogenesis, aging and other diseases. The 7,8-dihydro -8-oxo-guanine (8-oxoG or 8-OH-G) lesion is a major stable product of oxidative damage and has the most deleterious effects because it can mispair with adenine during DNA replication. MutY and MutY homolog (MYH) adenine glycosylases remove misincorporated adenines paired with template 8-oxoG and reduce G:C to T:A transversions, the mutagenic effects of 8-oxoG. This project will investigate on three aspects. (1) Structure-function of E. coli MutY. Because the C-terminal domain of MutY plays an important role in the recognition of 8-oxoG lesions, mutants at this domain will be constructed. A clamp model for the substrate recognition will be tested. (2) Interactions between MutY and S. pombe MYH (SpMYH) with enzymes involved in the base excision repair (BER) pathway. This study is based on the notion that BER must be a highly coordinated process to avoid releasing toxic intermediates. The mechanism of how MutY-protein interactions govern the efficiency and orchestra of the repair process will be investigated. To test the biological significance of these interactions, mutants of MutY and SpMYH that lack protein-protein interactions will be expressed in the mutY or SpMYH strains and the cell's phenotype will be examined. (3) Interaction and coordination of MutY (MYH) pathway with mismatch repair pathway, checkpoint pathway, and DNA replication.
This aim will study the interplay among MutY (MYH), MutS (MSH), beta subunit of DNA Pollll (PCNA), checkpoint proteins Husl/Radl/Rad9, and single-stranded DNA binding protein. Particularly, alterations in protein-protein interactions under oxidative stress will be investigated. These studies should advance our understanding of the role of DNA repair in genome stability and tumor susceptibility.
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