Nucleotide Excision Repair: From Recognition to Incision

核苷酸切除修复：从识别到切口

• 批准号: 6766181
• 负责人: CAROLINE F KISKER
• 金额: $ 29.87万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766181
• 关键词: DNA damage; DNA repair; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; binding sites; chromatography; conformation; crystallization; endodeoxyribonuclease; enzyme complex; nucleic acid sequence; nucleic acid structure; nucleotides; polymerase chain reaction; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): Maintenance of the correct genetic information is crucial for all living organisms. Mutations are the primary cause of hereditary diseases, as well as cancer, and may also be involved in aging. 80 to 90% of all human cancers are ultimately due to DNA damage. Different repair mechanisms have evolved to protect the genome. Nucleotide excision repair (NER) is well known for the removal of bulky DNA lesions and is unique in its versatility to repair a broad substrate range of DNA lesions. In humans, NER is the major repair mechanism to protect DNA from damage induced by ultraviolet light. The phenotypic consequences of defective genes involved in NER are apparent in three severe diseases: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy. The overall goals of this project are to understand the fundamental mechanisms of nucleotide excision repair by a series of studies involving the bacterial UvrABC NER machinery. In these studies specific proteins and complexes involved in nucleotide excision repair will be characterized by a combination of biochemical, crystallographic and molecular biology experiments. The studies described in this project will verify three hypotheses: (1) The bacterial NER protein UvrB recognizes damage by intercalating a b-hairpin between the DNA duplex and forms tight interactions with the undamaged strand only after formation of the pre-incision complex. (2) The lesion containing strand is mainly held in place by base stacking interactions and is freely accessible for recognition by UvrC, the endonuclease, which is responsible for the excision process. (3) A conformational change takes place in either UvrB and/or UvrC to allow the sequential process in which 3' incision precedes 5' incision. The proposed studies are divided into four specific aims: (1) Characterization of the UvrA/UvrB interaction and identification of DNA binding sites on UvrB. (2) The pre-incision complex: Structural characterization of the UvrB.DNA pre-incision complex. (3) Characterization of UvrC in the absence of UvrB and DNA: Determination of the three-dimensional structure of UvrC prior to binding to UvrB and DNA. (4) Analysis of the UvrB.UvrC.DNA complex: The proposed studies will delineate the roles of the individual proteins and their complexes formed in the process of NER.

描述（由申请人提供）：维持正确的遗传信息对于所有生物体至关重要。突变是遗传性疾病和癌症的主要原因，也可能与衰老有关。 80% 到 90% 的人类癌症最终是由于 DNA 损伤造成的。已经进化出不同的修复机制来保护基因组。核苷酸切除修复 (NER) 因去除大量 DNA 损伤而闻名，其独特之处在于其修复广泛底物范围的 DNA 损伤的多功能性。在人类中，NER 是保护 DNA 免受紫外线损伤的主要修复机制。 NER 相关缺陷基因的表型后果在三种严重疾病中很明显：着色性干皮病、科凯恩综合征和毛发硫营养不良。该项目的总体目标是通过一系列涉及细菌 UvrABC NER 机制的研究来了解核苷酸切除修复的基本机制。在这些研究中，将通过生化、晶体学和分子生物学实验的结合来表征参与核苷酸切除修复的特定蛋白质和复合物。该项目描述的研究将验证三个假设：（1）细菌 NER 蛋白 UvrB 通过在 DNA 双链体之间插入 b 发夹来识别损伤，并仅在形成预切割复合物后与未损伤的链形成紧密的相互作用。 (2) 含有损伤的链主要通过碱基堆积相互作用固定到位，并且可以自由地被 UvrC（核酸内切酶）识别，UvrC 负责切除过程。 (3) 在 UvrB 和/或 UvrC 中发生构象变化，以允许 3' 切口先于 5' 切口的顺序过程。拟议的研究分为四个具体目标： (1) UvrA/UvrB 相互作用的表征和 UvrB 上 DNA 结合位点的鉴定。 (2) 切口前复合物：UvrB.DNA 切口前复合物的结构表征。 (3)在没有UvrB和DNA的情况下UvrC的表征：在与UvrB和DNA结合之前确定UvrC的三维结构。 (4) UvrB.UvrC.DNA复合物的分析：拟议的研究将描述单个蛋白质及其在NER过程中形成的复合物的作用。