Defects in DNA repair pathways are associated with genome instability and carcinogenesis. DNA deamination results in conversion of adenosine to inosine, cytosine to uridine, and guanosine to xanthosine. Endonuclease V mediates an evolutionary conserved pathway involved in inosine repair. The long-term objective of this proiect is to elucidate the molecular mechanism underlying the endonuclease V-mediated repair pathway and understand its cellular functions in humans. The short-term goal of this proposal is to establish a cell-free repair system in Escherichia coli and explore its use in identifying cellular components involved in the endonuclease V-mediated DNA repair pathway. Substrates that can be used in the cell-free assays will be constructed. Protocols for the preparation of E. coli cell-free extracts will be developed. An in vitro assay system will be established to monitor endonuclease V-mediated repair activity. The repair patch created during the repair process will be determined. Biochemical approaches will be employed to identify downstream proteins in fractionated E. coli extracts that interact or displace endonuclease V. Assays will be devised to determine the biochemical functions of the identified proteins. The cell-free assay will be used to validate the roles of the downstream proteins in inosine repair, and identify additional proteins such as DNA polymerase required in this pathway. Insights gained from this study will provide a biochemical framework for investigation of human endonuclease V-mediated repair pathway.
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