Project 3: Conformational Studies of Adducted Oligodeoxynucleotides

项目3：加合寡脱氧核苷酸的构象研究

• 批准号: 7208783
• 负责人: Michael P Stone
• 金额: $ 35.49万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7208783
• 关键词: Archaea; DNA directed DNA polymerase; DNA repair; X ray crystallography; adduct; conformation; crosslink; gel mobility shift assay; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; oligonucleotides; site directed mutagenesis

The adduction of DNA by the environmentally and endogenously produced genotoxins vinyl chloride, acrolein, crotonaldehyde, and 4-hydroxynonenal (4-HNE), yields /nte/strand crosslinks, /nfrastrand crosslinks, DNA-protein conjugates, and regioisomeric mono-adducts. These chemicals likely contribute to background levels of inter- and /nfrastrand crosslinks and DNA-protein crosslinks in human cells. An inability to repair genomic damage correlates with human disease?e.g., cancer, premature aging, fatty liver disease, and atherosclerosis. Using materials prepared by Project 1 and the DNA Synthesis Core, this project will utilize NMR and crystallography to obtain high resolution structural data for DNA modified with these bis-electrophiles, to delineate the underlying structural basis for their mutagenicity and cytotoxicity. It will be determined how formation of /nterstrand crosslinks depends upon the identity and stereochemistry of substituents at C6 of proximal 1,N2-dG enal adducts. Using reduced crosslinks of the 6R- and 6S crotonaldehyde adducts it will be determined if the 6S crosslink creates a greater structural perturbation to DNA than does the 6R crosslink. The (6S,8R,11S) crosslink of the 4-HNE adduct possessing the same absolute stereochemistry as the 6R-crotonaldehyde crosslink will be characterized as to its chemistry. Work with reduced peptide-DNA crosslinks will focus on the conformation of the glycosyl torsion angle, which will orient the peptide in the minor vs. major groove. Our work will be correlated with that in Project 2 designed to understand crosslink repair. The chemistry of intrastrand crosslinks will be determined via incorporation of NMR-active isotopes. Using saturated analogs of these crosslinks, it will be determined if the intervening T in the 5'-GTX-3' sequence extrudes from the duplex. Residual dipolar coupling measurements in partially oriented samples, and gel electrophoretic mobility assays, will probe crosslink-induced DNA bending. The capacity of the Sulfolobus solfataricus Dpo4 polymerase to accommodate these crosslinks will be examined with crystallography of binary (Pol + DNA) and ternary (Pol + DNA + dNTP) complexes. The results will be correlated with site-specific mutagenesis experiments in Project 2. NMR studies will examine the hypothesis that all but one of the stereoisomeric 4-HNE adducts exist largely as 1 ,N2-dG adducts in the syn conformation about the glycosyl bond. Likewise, N1-dA and N3-dC adducts, and their N1-dl and N3-dU deamination products, may exist in the syn conformation about the glycosyl bond. Crystallography involving binary and ternary complexes of human Pol-iota and Pol-kappa with modified primer-template complexes will probe the sequential bypass of enal adducts by these polymerases, as observed by Project 2. It will be determined if hPol-iota exploits the syn conformations of these adducts for bypass. The role of hPol-kappa in positioning the primer 3'-OH to catalyze extension following insertion by hpol-iota will be determined. The potential for strand slippage during bypass of these lesions by Pol-eta, leading to frameshifts, will be determined.

通过环境和内源性产生的基因毒素乙烯基的氯化物的DNA的内收， 丙烯醛，crotonaldehede和4-羟基烯烯（4-hne）产生 /nte /strand crosslinks， /nfrastrand 交叉链接，DNA-蛋白结合物和区域异构单元。这些化学物质可能有助于 人类细胞中交联和DNA-蛋白交联的背景水平。无法 修复基因组损伤与人类疾病相关吗？例如，癌症，过早衰老，脂肪肝病， 和动脉粥样硬化。使用项目1和DNA合成核心准备的材料，该项目将 利用NMR和晶体学获取使用这些双电噬细胞修饰的DNA的高分辨率结构数据， 描绘其诱变性和细胞毒性的基本结构基础。这将是 确定 /nterstrand交联的形成如何取决于 近端1，N2-DG依赖加合物的C6的取代基。使用6R和6s的交联降低 双醛加合物是否会确定6S交叉链接会产生更大的结构扰动 DNA比6R交叉链接。 4-HNE加合物具有相同的（6s，8r，11s）的交联 绝对立体化学作为6R-甲醛交叉链接的化学化学将以其化学为特征。工作 随着肽-DNA交联的减少，将集中于糖基扭转角的构象，将 将肽与小凹槽与主要凹槽定向。我们的工作将与旨在的项目2中的工作相关 了解交联维修。汇入交联的化学将通过合并确定 NMR活性同位素。使用这些交联的饱和类似物，将确定是否中间t 在5'-GTX-3'中，序列从双链体挤出。部分的残留偶极耦合测量 面向的样品和凝胶电泳迁移率分析将探测交联引起的DNA弯曲。这 将检查可容纳这些交叉链接的磺胺溶液溶液溶液的能力 带有二元（POL + DNA）和三元（POL + DNA + DNTP）复合物的晶体学。结果将是 与项目2中的位点特异性诱变实验相关。NMR研究将研究假设 除了一个立体异构体4-HNE加合物中，其余所有除了SYN中的N2-DG加合物中都存在 关于糖基键的构象。同样，N1-DA和N3-DC加合物以及它们的N1-DL和N3-DU 脱氨基产物可能存在于涉及糖基键的同构中。涉及晶体学 带有修饰的引物 - 板络合物的人类Pol-iota和Pol-kappa的二元和三元络合物将探测 如项目2所观察到的这些聚合酶的连续绕过磁化加合物的顺序旁路。 如果HPOL-iota利用这些加合物的同步构象来绕过。 hpol-kappa在定位 将确定HPOL-iota插入后的底漆3'-OH催化延伸。链的潜力 将确定POL-ETA的这些病变途中的滑动，并确定导致翻新的。