Project 3: Conformational Studies of Adducted Oligodeoxynucleotides

环境和内源性产生的基因毒素氯乙烯对DNA的加合作用， 丙烯醛、巴豆醛和4-羟基壬烯醛（4-HNE）产生nte/strand交联， 交联、DNA-蛋白质缀合物和区域异构单加合物。这些化学物质可能有助于 人细胞中链间和链内交联以及DNA-蛋白质交联的背景水平。无力 修复与人类疾病相关的基因组损伤例如，在一个实施例中，癌症，早衰，脂肪肝， 和动脉粥样硬化。利用项目1和DNA合成核心准备的材料，该项目将 利用NMR和晶体学获得用这些双亲电体修饰的DNA的高分辨率结构数据， 描述其致突变性和细胞毒性的潜在结构基础。将 确定了链间交联的形成如何取决于以下的特性和立体化学： 邻位1，N2-dG烯醛加合物的C6取代基。使用6 R-和6S的减少的交联 巴豆醛加合物，将确定6S交联是否产生更大的结构扰动， DNA比6 R交联。具有相同结构的4-HNE加合物的（6S，8 R，11 S）交联 绝对立体化学，因为6 R-巴豆醛交联将表征其化学性质。工作 减少肽-DNA交联将集中在糖基扭转角的构象上，这将 将肽定位在小沟与大沟中。我们的工作将与项目2中的工作相关联， 了解交联修复。链内交联的化学性质将通过掺入 NMR活性同位素。使用这些交联的饱和类似物，将确定插入T 在5 '-GTX-3'序列中的一个或多个的双链体从双链体挤出。部分中的残余偶极耦合测量 定向样品和凝胶电泳迁移率测定将探测交联诱导的DNA弯曲。的 将检查硫磺硫化叶菌Dpo 4聚合酶容纳这些交联的能力 与二元（Pol + DNA）和三元（Pol + DNA + dNTP）复合物的晶体学。结果将 与项目2中的位点特异性诱变实验相关。核磁共振研究将检验这一假设 除了一种以外，所有的立体异构的4-HNE加合物主要以1，N2-dG加合物的形式存在于顺式异构体中。 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，可以以关于糖基键的顺式构象存在。晶体学涉及 人Pol-iota和Pol-kappa与修饰的引物-模板复合物的二元和三元复合物将探测 如项目2所观察到的，通过这些聚合酶顺序绕过烯醛加合物。将确定 如果hPol-iota利用这些加合物的顺式构象用于旁路。hPol-kappa在肿瘤定位中的作用 将确定引物3 ′-OH在插入hpol-i后催化延伸。潜在的股 将确定Pol-eta绕过这些病变期间的滑动，导致移码。

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.