Formation and Fate of Oxidative DNA-Protein Crosslinks

描述（由申请人提供）：2012年美国报告了约160万例新发癌症病例，其中约三分之一预计将因治疗疾病的并发症而导致死亡。癌症的复杂性和侵袭性取决于突变的积累。有越来越多的文献将基因组不稳定性与致癌联系起来，其中很多关注氧化损伤。基因组不断受到各种内源性和外源性损伤的攻击，最值得注意的是那些产生活性氧（ROS）的损伤。沿着其他病变，暴露于这些物质会产生氧化脱碱基位点。一个这样的例子，由C1 '氧化产生的2-脱氧核糖内酯（dL）可以引发DNA-蛋白质交联（DPC）与修复酶和其他蛋白质的形成。DNA氧化损伤的主要修复机制是碱基切除DNA修复途径（BER）。在尝试通过DNA聚合酶酶切（Polymerase）对dL进行BER期间，酶的5'-脱氧核糖-5-磷酸（5'dRp）裂解酶活性试图切除病变，但被捕获，导致DPC形成。其他DNA裂解酶（一些DNA糖基化酶、DNA聚合酶γ和λ以及Ku蛋白）也被dL残基共价捕获。然而，细胞如何修复这些DPC仍然是未知的。该提案旨在了解DL介导的DPC的诱导和去除动力学，并揭示这些病变是否在哺乳动物细胞中被主动修复。目的1的目标提出开发或调整灵敏的生物化学测定，其将使得能够在体内研究DL-聚乙烯DPC。目的2将确定使用dL诱导剂在细胞中形成Polymorphic DPC所需的条件，并将跟踪它们从基因组中的去除动力学。最后，目标3将研究是否dL-Polymorphic DPC被主动删除修复过程涉及同源重组，核苷酸切除修复或一些组合，可能涉及蛋白水解作为修复的先决条件。我预计这些研究的结果将提供有关DPC对DNA损伤的贡献的新信息，并开辟新的研究途径，以了解它们与人类致癌作用的关系。

DESCRIPTION (provided by applicant): About 1.6 million new cancer cases were reported for 2012 in the U.S.A, of which about one-third were expected to result in death due to complications in treating the disease. The complexity and aggressiveness of cancers depends on the accumulation of mutations. There is a growing body of literature linking genomic instability to carcinogenesis, with much focus on oxidative damage. The genome is constantly under assault by a variety of endogenous and exogenous insults, most notably those that generate reactive oxygen species (ROS). Along with other lesions, exposure to these agents produces oxidized abasic sites. One such example, 2-deoxyribonolactone (dL), which results from C1' oxidation, can instigate the formation of DNA-protein crosslinks (DPCs) with repair enzymes and other proteins. The principal repair mechanism for oxidative DNA damage is the base excision DNA repair pathway (BER). During attempted BER of dL by, DNA polymerase ß (Polß), the enzyme's 5'-deoxyribose-5-phosphate (5'dRp) lyase activity attempts to excise the lesion but becomes trapped leading to DPC formation. Other DNA lyase enzymes (some DNA glycosylases, DNA polymerases γ and λ, and the Ku proteins) also become covalently trapped by dL residues. However, it remains unknown how cells may repair these DPCs. This proposal seeks to understand the induction and removal kinetics of dL-mediated DPCs, and to uncover whether these lesions are actively repaired in mammalian cells. The goals of Aim 1 propose to develop or adapt a sensitive biochemical assay which will enable the study of dL-Polß DPCs in vivo. Aim 2 will identify the conditions required to form Polß DPCs in cells using dL-inducing agents, and their removal kinetics from the genome will be followed. Finally, Aim 3 will investigate whether dL-Polß DPCs are actively removed by repair processes involving homologous recombination, nucleotide excision repair or some combination that may involve proteolysis as a prerequisite for repair. I anticipate that the findings of these studies will provde new information on the contribution of DPCs to DNA damage and open new avenues of investigation to understand their relationship to human carcinogenesis.