Mechanistic Investigation of DNA Lesion Bypass

各种DNA损伤剂不断攻击细胞DNA并产生无数损伤。例如，顺铂与抑制细胞生长的细胞DNA形成许多交联。未修复的损伤阻断复制DNA聚合酶，但可以被最近发现的Y家族DNA聚合酶的成员绕过。在人类中，16种已鉴定的DNA聚合酶中有4种属于Y家族。Y家族聚合酶缺乏固有的校对活性，并且通常以高错误率催化DNA合成。Y家族DNA聚合酶对未修复损伤的这种易错复制与诱变有关。对于这个项目，古细菌酶Dpo 4被选择作为模型Y家族聚合酶。dpo 4含有四个结构域。我们的目标是了解Dpo4的这些结构域如何在DNA合成过程中在存在和不存在DNA损伤的情况下动态移动。DNA损伤旁路产物将被测序以揭示由易错Dpo 4产生的突变的身份。将确定Dpo 4、顺铂损伤的DNA和核苷酸（DNA“构建块”）的复合物的晶体结构。这个结构将揭示Dpo4如何复制受损的DNA。Dpo 4获得的结果可以应用于真核生物，因为Dpo 4的活性与其他Y家族聚合酶非常相似。此外，与DPO 4观察到的构象动力学和DNA合成之间的耦合可能描绘了DNA聚合酶一般如何起作用。除了科学意义外，该研究项目还将为俄亥俄州州立大学的三名研究生和几名本科生提供机会，接受先进酶学领域的重要科学培训。为了培养他们的实验技能，这个研究项目将为他们提供设计和测试科学假设以及理解科学发现的本质的智力工具。

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.