T4 Endonuclease V Structure-Function Analysis

描述（由申请人提供）：已证明短波紫外线暴露是非黑色素瘤皮肤癌的致病因素，也是黑色素瘤的一个强风险因素。虽然人类细胞仅使用核苷酸切除修复来修复UV诱导的二嘧啶DNA光产物，但其他生物体通过催化嘧啶二聚体5'碱基处的切割的DNA糖基化酶启动碱基切除修复途径。发展对酶的功能的理解是至关重要的，因为T4嘧啶二聚体糖基化酶（T4-Pdg）正在用于人体临床试验。尽管在着色性干皮病患者上局部递送野生型T4-Pdg已证明在癌症减少中的功效，但迄今为止使用野生型哺乳动物细胞的所有研究揭示，T4-Pdg导致UV后存活率降低，而不是增加。假设T4-Pdg切割DNA结构域内所有二聚体位点的能力导致细胞毒性双链断裂，其中二聚体在互补链中非常接近。因此，假设失去切割簇中二聚体能力的T4-Pdg形式将增强修复并减少诱变，而不产生细胞毒性双链断裂。为了实现这一目标，提出将T4-Pdg工程化以在DNA弯曲和核苷酸翻转的预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物和切割成簇的二聚体的能力降低。这些研究将指导我们最近确定的共晶结构的T4-Pdg共价捕获作为还原亚胺中间体在双链体DNA的脱碱基位点。这种结构揭示了实现活性复合物所必需的关键氨基酸，这些数据导致了一系列假设，这些假设涉及酶在此过程中的至少三个不同部分。利用从共晶结构中获得的知识，并考虑到增强野生型哺乳动物细胞中二聚体修复的挑战，提出了具体目标：1）对T4-Pdg突变体进行弯曲、翻转、催化和成簇的能力进行生物化学表征体外切口; 2）在角质形成细胞中表达对照和突变T4-Pdgs以确定对双链断裂形成、存活和诱变的影响;和3）激活线粒体中UV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.