Formation and Fate of Oxidative DNA-Protein Crosslinks

描述(申请人提供)：2012年美国报告的新增癌症病例约为160万例，其中约三分之一预计会因治疗该疾病的并发症而死亡。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与癌症发生联系起来，重点放在氧化损伤上。基因组不断受到各种内源性和外源性侮辱的攻击，最明显的是那些产生活性氧物种(ROS)的侮辱。与其他损害一样，暴露在这些制剂中会产生氧化的碱性部位。一个这样的例子，2-脱氧核糖内酯(DL)，它是由c1‘氧化产生的，可以与修复酶和其他蛋白质形成DNA-蛋白质交联链(DPC)。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dL by，DNA聚合酶(Pol？)的BER期间，该酶的5‘-脱氧核糖-5-磷酸(5’DRP)裂解酶活性试图切除病变，但被困住，导致DPC形成。其他dna裂解酶(一些dna糖基酶，dna聚合酶γ和λ，以及Ku蛋白)也会被dL残基共价捕获。然而，目前尚不清楚细胞如何修复这些DPC。这项建议试图了解dL介导的DPC的诱导和去除动力学，并揭示这些损伤是否在哺乳动物细胞中得到积极修复。目标1的目标是开发或调整一种灵敏的生化分析方法，以便能够在体内研究dL-PolçDPC。目标2将确定使用dL诱导剂在细胞中形成PolçDPC所需的条件，并将跟踪它们从基因组中移除的动力学。最后，目标3将调查dL-PolçDPC是否被修复过程主动移除，修复过程涉及同源重组、核苷酸切除修复或作为修复先决条件的可能涉及蛋白质分解的某种组合。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，以了解它们与人类癌症发生的关系。

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.