Mechanistic Investigation of DNA Lesion Bypass

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Various DNA-damaging agents continually attack cellular DNA and generate a myriad of lesions. For example, cisplatin forms numerous cross-links with cellular DNA which inhibits cell growth. Unrepaired lesions block replicative DNA polymerases, but can be bypassed by members of the recently discovered Y-family DNA polymerases. In humans, four of the sixteen identified DNA polymerases are in the Y-family. The Y-family polymerases lack intrinsic proof-reading activity and often catalyze DNA synthesis with a high error rate. Such error-prone replication of unrepaired lesions by the Y-family DNA polymerases has been linked to mutagenesis. For this project, an archaeal enzyme Dpo4, is chosen as a model Y-family polymerase. Dpo4 contains four structural domains. The goal is to understand how these structural domains of Dpo4 dynamically move during DNA synthesis in the presence and absence of a DNA lesion. The DNA lesion bypass products will be sequenced to reveal the identity of mutations produced by error-prone Dpo4. The crystal structure of a complex of Dpo4, cisplatin-damaged DNA, and a nucleotide (DNA "building block") will be determined. This structure will reveal how Dpo4 replicates damaged DNA. The results obtained with Dpo4 can be applied to eukaryotic counterparts since the activity of Dpo4 closely mimics other Y-family polymerases. Furthermore, the coupling between conformational dynamics and DNA synthesis observed with Dpo4 likely portrays how a DNA polymerase functions in general.In addition to scientific importance, the research project will also offer three graduate students and a few undergraduate students at The Ohio State University opportunities to receive important scientific training in the field of advanced enzymology. To nurture their experimental skills, this research project will provide them intellectual tools for designing and testing scientific hypotheses and for understanding the nature of scientific discovery.