T4 Endonuclease V Structure-Function Analysis

T4 核酸内切酶 V 结构功能分析

基本信息

批准号：
7911342
负责人：
R. Stephen Lloyd
金额：
$ 33.1万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-04 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911342
关键词：
Actinic keratosis Active Sites Amino Acids Architecture Base Excision Repairs Binding Biological Catalysis Cells Clinical Trials Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Sequence Rearrangement DNA glycosylase DNA photoproducts Data Engineering Enzymes Exposure to Fibroblasts Frequencies Goals Human Imines In Vitro Investigation Knowledge Lesion Malignant Neoplasms Mammalian Cell Mitochondria Mitochondrial DNA Movement Mutagenesis Mutation Nucleotide Excision Repair Nucleotides Organism Pathway interactions Patients Process Pyrimidine Dimers Reaction Residual state Resistance Risk Factors Role Series Short Waves Side Site Skin Cancer Skin Carcinoma Structure Surgical incisions T4 Endonuclease V Testing Ultraviolet Rays United States Xeroderma Pigmentosum base cancer diagnosis carcinogenesis cytotoxic cytotoxicity dimer insight keratinocyte melanoma molecular dynamics mutant repaired tumor

项目摘要

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.

描述（由申请人提供）：已证明暴露于短波紫外线是非甲状腺瘤皮肤癌的因果因素，是黑色素瘤中强烈的危险因素。虽然人类细胞仅使用核苷酸切除修复来修复紫外二吡啶DNA光生型，但其他生物会通过DNA糖基化酶在嘧啶二聚体的5'碱基上催化切口的DNA糖基化酶启动碱基切除修复途径。由于T4嘧啶二聚体糖基酶（T4-PDG）在人类临床试验中使用，因此对酶的功能有所了解至关重要。尽管野生型T4-PDG局部递送在静脉表色素患者上表现出在癌症降低方面的功效，但迄今使用野生型哺乳动物细胞进行的所有研究表明，T4-PDG会导致uv后生存率降低，而不是紫外线后的存活率升高。假设T4-PDG切除DNA结构域内所有二聚体位点的能力会导致细胞毒性双链断裂，其中二聚体在互补链中处于紧密的距离。因此，假设失去切口二聚体能力的T4-PDG形式将增强修复并减少诱变，而不会产生细胞毒性的双链断裂。为了实现这一目标，建议设计T4-PDG在DNA弯曲和核苷酸翻转的催化步骤中的效率较低，其净结果是这些改变的酶在簇中形成Michaelis Complex的能力降低了簇中的切口和切口二聚体。这些研究将以我们最近确定T4-PDG的共晶结构的指导，将其作为减少的双杆DNA的无碱性位点的亚胺中间体共价捕获。该结构揭示了实现活动复合物所需的关键氨基酸，这些数据导致了一系列假设，这些假设在此过程中至少暗示了该酶的至少三个不同部分。使用源自共晶体结构的知识，并考虑到增强野生型哺乳动物细胞中二聚体修复的挑战，提出了特定的目的，提出了1）对T4-PDG的生物化学表征T4-PDG的突变体，以执行弯曲，翻转，翻转，催化和聚集的刺激。 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，存活和诱变的影响； 3）激活线粒体中紫外线生产的碱基切除修复，并确定二聚体在细胞毒性中的作用。