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

许多DNA损伤剂不断攻击细胞DNA，并产生各种损伤。没有修复的损伤会阻止复制的DNA聚合酶，但可以被新型Y家族DNA聚合酶的成员绕过。Y-家族聚合酶缺乏固有的校对活性，经常催化DNA合成，错误率很高。Y家族聚合酶对病变的这种错误复制有助于自发突变的产生；在人类中，这种突变过程已知会影响癌基因和肿瘤抑制基因。在本项目中，来自Sulfolobus Sulfararicus的古生菌酶Dpo4被选为Y家族的典型聚合酶。我们的目标是了解Dpo4如何复制未受损和受损的DNA。这些结果具有广泛的适用性，因为从细菌到人类的所有生物都含有Y-家族聚合酶。基于极快激光的动态技术(快到飞秒)将被用来研究DPO4、DNA和进入的核苷酸(DNA“积木”)之间的相互作用。将确定DPO4、顺铂损伤的DNA和核苷酸的复合体的晶体结构。顺铂是一种常用的抗癌药物，其形成的DNA加合物的结构已经很好地建立了。晶体结构将揭示DPO4如何与受损的DNA相互作用并复制。该研究项目不仅将提供对Y家族聚合酶绕过DNA损伤的全面了解，还将为俄亥俄州立大学的本科生和研究生提供在高级酶动力学和动力学领域接受重要科学培训的机会。

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.