MECHANISTIC STUDIES ON DNA REPAIR ENZYMES

DNA修复酶的机理研究

基本信息

批准号：
2180373
负责人：
TADHG P. BEGLEY
金额：
$ 15.04万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-07-01 至 1995-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2180373
关键词：
DNA damage DNA repair carbon carbon lyase chemical synthesis crystallization deuterium dimer enzyme mechanism enzyme substrate free radicals molecular cloning nucleic acid sequence oligonucleotides photochemistry spectrometry thymine ultraviolet radiation

项目摘要

Photodamage to DNA results in mutagenesis and cell death. In humans this damage can result in skin cancer. Repair of DNA photolesions is essential for the survival of living systems in sunlight and involves either an excision or in situ repair of the lesion. In this proposal, studies designed to elucidate the enzymatic mechanism of the in situ repair of the cyclobutane pyrimidine photodimer and the spore product are described. The major DNA damage caused by UV irradiation of bacteria is the cyclobutane thymine dimer. DNA photolyase catalyzes the in situ repair of this lesion in a light-dependent reaction. To conclude our mechanistic studies on this enzyme, we propose to further explore the use of iodomethyl substituted photodimers as probes for radical intermediates and to overexpress the Salmonella typhimurium photolyase for crystallographic studies. In contrast to bacterial DNA, UV irradiation of bacterial spores results in the formation of the spore product as the major lesion. We will develop a simple model system for the conversion of bis-thymines to spore product. We will develop a synthetic route to the dinucleotide spore product and incorporate this into short oligonucleotides to determine the minimum substrate required for the repair enzyme. we will explore the mechanism of spore product formation and repair using a combination of stereochemical, isotope effect and radical or carbanion trapping studies.

DNA对DNA的光损伤导致诱变和细胞死亡。在人类中损害会导致皮肤癌。 DNA光子的修复是对于阳光下生存系统的生存至关重要的，涉及切除或现场修复病变。在此提案中，旨在阐明原位的酶促机理的研究修复环丁烷嘧啶光二聚体和孢子产物是描述。细菌紫外线照射造成的主要DNA损伤是环丁烷胸腺二聚体。 DNA光溶酶催化原位修复在光依赖性反应中的病变。总结我们关于这种酶的机械研究，我们建议进一步探索使用碘甲基取代的光二聚体作为自由基中间体的探针并过表达鼠伤寒沙门氏菌光溶解酶的晶体学研究。与细菌DNA相反，细菌孢子的紫外线照射结果在形成孢子产物作为主要病变中。我们将开发一个简单的模型系统，用于将双胸腺胺转化为孢子产品。我们将开发一条通往二核苷酸孢子的合成途径产品并将其纳入短寡核苷酸以确定修复酶所需的最小基材。我们将探索孢子产物形成和维修的机制结合立体化学，同位素效应以及激进或碳纤维诱捕研究。