Nucleotide Excision Repair: From Recognition to Incision

描述（由申请人提供）：维护正确的遗传信息对所有生物都至关重要。突变是遗传疾病和癌症的主要原因，也可能与衰老有关。80%到90%的人类癌症最终都是由于DNA损伤。为了保护基因组，进化出了不同的修复机制。核苷酸切除修复（NER）以去除大体积DNA损伤而闻名，其多功能性修复广泛的DNA损伤是独一无二的。在人类中，NER是保护DNA免受紫外线损伤的主要修复机制。参与NER的缺陷基因的表型后果在三种严重疾病中是明显的：着色性干皮病、科凯恩综合征和毛硫营养不良。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，涉及核苷酸切除修复的特定蛋白质和复合物将通过生物化学，晶体学和分子生物学实验的结合来表征。本项目所描述的研究将验证三个假设：(1)细菌NER蛋白UvrB通过在DNA双链之间插入b-发夹识别损伤，并仅在形成切口前复合体后与未受损链形成紧密相互作用。(2)病变包含链主要通过碱基堆叠相互作用保持在原位，并且可以自由地被UvrC识别，UvrC是负责切除过程的内切酶。(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：

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.