T4 Endonuclease V Structure-Function Analysis

T4 核酸内切酶 V 结构功能分析

• 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
• 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

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）：暴露于短波紫外线已被证明是非黑色素瘤皮肤癌的致病因素，也是黑色素瘤的强烈危险因素。虽然人类细胞仅使用核苷酸切除修复来修复紫外线诱导的二嘧啶 DNA 光产物，但其他生物体通过 DNA 糖基化酶启动碱基切除修复途径，催化嘧啶二聚体 5' 碱基的切割。了解酶的功能至关重要，因为 T4 嘧啶二聚体糖基化酶 (T4-Pdg) 正在用于人体临床试验。尽管野生型 T4-Pdg 局部给药于色素性干皮病患者已被证明可有效减少癌症，但迄今为止使用野生型哺乳动物细胞进行的所有研究均表明，T4-Pdg 会导致紫外线后存活率下降，而不是增加。据推测，T4-Pdg 切割 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非常接近。因此，假设失去了切割簇中二聚体能力的 T4-Pdg 形式将增强修复并减少诱变，而不会产生细胞毒性双链断裂。为了实现这一目标，建议对 T4-Pdg 进行改造，使其在 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体和切割簇中二聚体的能力降低。这些研究将以我们最近确定的 T4-Pdg 共晶结构为指导，该 T4-Pdg 作为还原亚胺中间体共价捕获在双链 DNA 的脱碱基位点上。这种结构揭示了获得活性复合物所必需的关键氨基酸，并且这些数据导致了一系列假设，这些假设暗示了该过程中酶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，提出的具体目标是：1）生化表征T4-Pdg突变体在体外进行弯曲、翻转、催化和聚集切口的能力； 2) 在角质形成细胞中表达对照和突变体T4-Pdgs，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。

项目成果

Mutation of tryptophan 128 in T4 endonuclease V does not affect glycosylase or abasic site lyase activity.

T4 核酸内切酶 V 中色氨酸 128 的突变不会影响糖基酶或脱碱基位点裂解酶活性。

• DOI: 10.1021/bi00196a021
• 发表时间: 1994
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Latham,KA;Carmical,JR;Lloyd,RS
• 通讯作者: Lloyd,RS

T4 endonuclease V exists in solution as a monomer and binds to target sites as a monomer.

T4 核酸内切酶 V 作为单体存在于溶液中，并作为单体与靶位点结合。

• DOI: 10.1016/0167-4838(95)00224-3
• 发表时间: 1996
• 期刊: Biochimica et biophysica acta
• 作者: Latham,KA;Rajendran,S;Carmical,JR;Lee,JC;Lloyd,RS
• 通讯作者: Lloyd,RS

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair.

蛋白质-DNA 相互作用中促进扩散的生物学意义。

• 发表时间: 1990
• 期刊: The Journal of biological chemistry
• 作者: Dowd,DR;Lloyd,RS
• 通讯作者: Lloyd,RS

Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase.

t4 嘧啶二聚体 DNA 糖基化酶解偶联核苷酸翻转和 DNA 弯曲。

• DOI: 10.1021/bi060802s
• 发表时间: 2006
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Walker,RandallK;McCullough,AmandaK;Lloyd,RStephen
• 通讯作者: Lloyd,RStephen

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

紫外线诱导的环丁烷嘧啶二聚体对真核拓扑异构酶 II 的抑制。

• 发表时间: 1991
• 期刊: The Journal of biological chemistry
• 作者: Corbett,AH;Zechiedrich,EL;Lloyd,RS;Osheroff,N
• 通讯作者: Osheroff,N