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

职业：Y 家族聚合酶的动力学、动态和结构功能关系研究

• 批准号: 0447899
• 负责人: Zucai Suo
• 金额: $ 70万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0447899&HistoricalAwards=false
• 关键词: CAREER; Kinetic; Dynamic; Structure; Function

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.

许多DNA损伤剂持续攻击细胞DNA并产生各种损伤。未修复的损伤阻断了复制型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，并且通常以高错误率催化DNA合成。Y家族聚合酶的这种易出错的病变复制有助于自发突变的产生;在人类中，已知这种突变过程会影响癌基因和肿瘤抑制基因。对于该项目，选择来自Sulfolobus sulfataricus的古菌酶Dpo 4作为模型Y家族聚合酶。目的是了解Dpo 4如何复制未受损和受损的DNA。结果是广泛适用的，因为从细菌到人类的所有生物体都含有Y家族聚合酶。基于极快激光的动态技术（快至飞秒）将用于研究Dpo 4、DNA和进入的核苷酸（DNA“构建块”）之间的相互作用。将测定Dpo 4、顺铂损伤的DNA和核苷酸的复合物的晶体结构。顺铂是一种常用的抗癌药物，其形成的DNA加合物的结构已得到很好的确定。晶体结构将揭示Dpo 4如何与受损DNA相互作用并复制受损DNA。该研究项目不仅将提供一个全面的了解DNA病变旁路的Y-家族聚合酶，但也将提供本科生和研究生在俄亥俄州州立大学的机会，接受重要的科学培训，在先进的酶动力学和动力学领域。