T4 Endonuclease V Structure-Function Analysis

描述(由申请人提供)：暴露在短波紫外线下已被证明是非黑色素瘤皮肤癌的原因因素，也是黑色素瘤的强烈危险因素。虽然人类细胞只使用核苷酸切除修复来修复紫外线诱导的二嘧啶DNA光产物，但其他生物通过DNA糖基酶启动碱基切除修复途径，该酶催化在嘧啶二聚体的5‘碱基上切割。由于T4嘧啶二聚体糖基酶(T4-PDG)正被用于人体临床试验，因此了解酶的功能至关重要。尽管外用野生型T4-PDG治疗色素性干皮病患者已显示出减少癌症的效果，但迄今为止使用野生型哺乳动物细胞的所有研究表明，T4-PDG导致紫外线照射后存活率降低，而不是增加。假设T4-PDG能够切割DNA结构域中的所有二聚体位置，导致在互补链中二聚体非常接近的地方发生细胞毒性双链断裂。因此，假设失去了切割簇内二聚体能力的T4-PDG形式将在不产生细胞毒性双链断裂的情况下增强修复和减少突变。为了实现这一目标，有人提议将T4-PDG改造成在DNA弯曲和核苷酸翻转的前催化步骤中效率较低，最终结果是这些改变的酶形成米氏复合体和切割成簇的二聚体的能力降低。这些研究将以我们最近确定的T4-PDG的共晶结构为指导，T4-PDG作为还原的亚胺中间体被捕获在双链DNA的碱性位置。这种结构揭示了实现活性复合体所必需的关键氨基酸，这些数据导致了一系列假说，这些假说至少涉及酶的三个不同部分。利用协晶结构方面的知识，并鉴于加强野生型哺乳动物细胞中的二聚体修复所面临的挑战，我们提出了以下具体目标：1)对突变体T4-PDG在体外进行弯曲、翻转、催化和丛生切割的能力进行生化表征；2)在角质形成细胞中表达对照和突变体T4-PDG，以确定对双链断裂形成、存活和突变的影响；以及3)激活线粒体紫外光产物的碱基切除修复，并确定二聚体在细胞毒性中的作用。

DESCRIPTION (provided by applicant): Exposure to short wave ultraviolet light has been demonstrated to be the causal factor in nonmelanoma skin cancers and a strong risk factor in melanomas. While human cells only use nucleotide excision repair to repair the UV-induced dipyrimidine DNA photoproducts, other organisms initiate the base excision repair pathway by DNA glycosylases that catalyze incision at the 5' base of pyrimidine dimers. Developing an understanding of the function of enzymes is critical, since T4 pyrimidine dimer glycosylase (T4-Pdg) is being used in human clinical trials. Although topical delivery of wild-type T4-Pdg on xeroderma pigmentosum patients has demonstrated efficacy in cancer reduction, all investigations to date using wild-type mammalian cells reveal that T4-Pdg results in decreased, rather than increased survival after UV. It is hypothesized that the ability of T4-Pdg to incise all dimer sites within DNA domains leads to cytotoxic double-strand breaks where dimers are in close proximity in complementary strands. Thus, it is hypothesized that forms of T4-Pdg that have lost the ability to incise dimers in clusters will enhance repair and decrease mutagenesis without creating cytotoxic double-strand breaks. To accomplish this goal, it is proposed to engineer T4-Pdg to be less efficient in the precatalytic steps of DNA bending and nucleotide flipping, with the net result being a decrease in the ability of these altered enzymes to form a Michaelis complex and incise dimers in clusters. These studies will be guided by our recent determination of the cocrystal structure of T4-Pdg covalently trapped as a reduced imine intermediate at an abasic site in duplex DNA. This structure reveals key amino acids necessary for achieving an active complex, and these data have led to a series of hypotheses that implicate at least three different portions of the enzyme in this process. Using the knowledge derived from the cocrystal structure, and given the challenges of enhancing dimer repair in wild-type mammalian cells, Specific Aims are proposed to 1) biochemically characterize mutants of T4-Pdg in their ability to carry out bending, flipping, catalysis, and clustered incisions in vitro; 2) express control and mutant T4-Pdgs in keratinocytes to determine effects on double-strand break formation, survival, and mutagenesis; and 3) activate base excision repair of UV-photoproducts in mitochondria and determine the role of dimers in cytotoxicity.