CAREER: Kinetic, Dynamic, and Structure-Function Relationship Studies of a Y-family Polymerase

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Numerous DNA-damaging agents continually attack cellular DNA and generate a variety of lesions. Lesions that are not repaired block replicative DNA polymerases, but can be bypassed by members of the novel Y-family DNA polymerases. The Y-family polymerases lack intrinsic proof-reading activity and often catalyze DNA synthesis with a high error rate. Such error-prone replication of lesions by Y-family polymerases contributes to the generation of spontaneous mutations; in human this mutational process is known to affect oncogenes and tumor suppressor genes. For this project, an archaeal enzyme Dpo4, from Sulfolobus sulfataricus is chosen as a model Y-family polymerase. The goal is to understand how Dpo4 copies undamaged and damaged DNA. The results are broadly applicable since all organisms from bacteria to humans contain Y-family polymerases. Extremely fast laser based dynamic techniques (as fast as femtoseconds) will be used to study the interactions between Dpo4, DNA, and an incoming nucleotide (DNA "building block"). The crystal structure of a complex of Dpo4, cisplatin-damaged DNA, and a nucleotide will be determined. Cisplatin is a frequently used anti-cancer drug and the structure of the DNA adduct that forms is well established. The crystal structure will reveal how Dpo4 interacts with and replicates damaged DNA. The research project will not only provide a comprehensive understanding of DNA lesion bypass by a Y-family polymerase, but will also offer undergraduate and graduate students at the Ohio State University opportunities to receive important scientific training in the field of advanced enzyme kinetics and dynamics.