Nucleosomes Modulate DNA Interstrand Crosslink Repair

核小体调节 DNA 链间交联修复

• 批准号: 6614836
• 负责人: Muriel W Lambert
• 金额: $ 29.55万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6614836
• 关键词: DNA damage; DNA repair; SDS polyacrylamide gel electrophoresis; crosslink; endonuclease; enzyme activity; gel mobility shift assay; molecular shape; nucleic acid structure; nucleosomes; phosphoester ligase; protein purification; protein structure function; site directed mutagenesis; western blottings

DESCRIPTION (provided by applicant): Chromatin structure plays a critical role in modulation of cellular response to DNA damage. The mechanism utilized by repair proteins to locate target sites on damaged nucleosomal DNA is critical to understanding these repair processes. The present proposal will examine the effect of nucleosome structure on the ability of human repair proteins to interact with and incise DNA containing interstrand cross-links, produced by potentially mutagenic and carcinogenic environmental agents such as psoralen plus UVA light, and to ascertain whether there is a relationship between the processive mechanism of action shown by the endonucleases involved in this process and their ability to incise damaged nucleosomal DNA. We have isolated a protein complex from the nuclei of normal human cells that contains all the proteins needed for recognition and incision of DNA containing interstrand cross-links. In cells from patients with the cancer-prone, repair deficient genetic disease, xeroderma pigmentosum, complementation group A (XPA), this same complex has normal levels of activity on cross-linked naked DNA but is defective in ability to incise damaged nucleosomal DNA. This defect correlates with the distributive mechanism of action utilized by the endonucleases present in the XPA complex, in contrast to the processive mechanism of action of the normal endo-nucleases, and is related to the loss of ability of the XPA protein to act as a processivity factor. The present proposal will focus on this new role of XPA as a processivity factor and on which domain in XPA is responsible for its mechanism of action. Site-directed mutagenesis will be used to create selected mutations in XPA. Recombinant mutant proteins will be produced and examined to ascertain which domain(s) is needed for its action as a processivity factor on damaged naked DNA and the importance of this domain for its action on damaged nucleosomal DNA. DNA substrates will be constructed that contain a positioned nucleosome and a site-specific 4,5',8-trimethyl-psoralen (TMP) interstrand cross-link in either the core or linker region. This will allow evaluation of the effect of nucleosome structure and cross-link location on the influence of the mutant and wild type XPA proteins on incisions produced by the normal and XPA complexes on damaged DNA. Whether defects in ability of the XPA complex to incise damaged nucleosomal DNA can be corrected by the wild type or mutant XPA proteins will also be examined. These studies will provide important insights into the complex mechanism by which interstrand cross-links are repaired in nucleosomal DNA, the importance of a processive mechanism of action by repair proteins on damaged nucleosomal DNA, and the severe consequences that occur in individuals when this mechanism is defective.

描述（由申请人提供）：染色质结构在调节细胞对DNA损伤的反应中起关键作用。修复蛋白在受损核小体DNA上定位靶位点的机制对于理解这些修复过程至关重要。本提案将研究核小体结构对人类修复蛋白与含有链间交联的DNA相互作用和切割DNA的能力的影响，所述链间交联由潜在的致突变和致癌环境因子如peptide + UVA光产生，并确定参与该过程的核酸内切酶所显示的进行性作用机制与它们切割受损细胞的能力之间是否存在关系，核小体DNA我们已经从正常人类细胞的细胞核中分离出一种蛋白质复合物，它含有识别和切割含有链间交联的DNA所需的所有蛋白质。在来自具有癌症倾向的、修复缺陷型遗传疾病、着色性干皮病、互补组A（XPA）的患者的细胞中，这种相同的复合物对交联的裸DNA具有正常水平的活性，但在切割受损的核小体DNA的能力上是有缺陷的。这种缺陷与XPA复合物中存在的核酸内切酶所利用的作用的分布机制相关，与正常核酸内切酶的进行性作用机制相反，并且与XPA蛋白作为持续合成因子的能力丧失有关。目前的建议将集中在XPA作为合成因子的新作用，以及XPA中的哪个域负责其作用机制。定点诱变将用于在XPA中产生选定的突变。重组突变蛋白将被生产和检查，以确定其作为受损裸DNA的持续合成因子的作用需要哪个结构域，以及该结构域对于其对受损核小体DNA的作用的重要性。将构建DNA底物，其含有定位的核小体和在核心或接头区中的位点特异性4，5 '，8-三甲基-戊二烯（TMP）链间交联。这将允许评估核小体结构和交联位置对突变型和野生型XPA蛋白对由正常和XPA复合物在受损DNA上产生的切口的影响的影响。还将检查XPA复合物切割受损核小体DNA的能力缺陷是否可以通过野生型或突变型XPA蛋白来校正。这些研究将提供重要的见解复杂的机制，其中链间交联修复在核小体DNA，修复蛋白对受损的核小体DNA的重要性，以及严重的后果，发生在个人时，这种机制是有缺陷的进行性机制的作用。