Nucleosomes Modulate DNA Interstrand Crosslink Repair

描述(由申请人提供)：染色质结构在调节细胞对DNA损伤的反应中起着关键作用。修复蛋白用来定位受损核小体DNA上的靶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潜在诱变和致癌环境物质如补骨脂素加UVA光产生的含有链间交叉连接的DNA相互作用和切割的能力的影响，并确定参与这一过程的内切酶所显示的过程作用机制与它们切割受损核小体DNA的能力之间是否存在关系。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体，该复合体包含识别和切割含有链间交联链的DNA所需的所有蛋白质。在易患癌症、修复缺陷的遗传病、着色性干皮病、补体A组(XPA)患者的细胞中，同样的复合体在交联的裸DNA上具有正常水平的活性，但切割受损核小体DNA的能力存在缺陷。这一缺陷与XPA复合体中存在的内切酶所利用的分布作用机制有关，而不是与正常内切酶的过程性作用机制相反，并与XPA蛋白失去作为加工性因子的能力有关。本提案将侧重于XPA作为过程因素的这一新作用，以及XPA中的哪个领域负责其作用机制。定点突变将被用来在XPA中产生选定的突变。重组突变蛋白将被生产和检测，以确定其作为损伤的裸DNA的处理因子所需的结构域(S)以及该结构域对其损伤的核小体DNA的作用的重要性。DNA底物将被构建成在核心或连接区包含定位的核小体和位点特异性的4，5‘，8-三甲基补骨脂素(TMP)链间交联物。这将使我们能够评估核小体结构和交联位置对突变型和野生型XPA蛋白对正常和XPA复合体在受损DNA上产生的切割的影响的影响。还将检查XPA复合体切割受损核小体DNA的能力缺陷是否能被野生型或突变型XPA蛋白纠正。这些研究将为核小体DNA链间交联修复的复杂机制，修复蛋白对受损核小体DNA的过程作用机制的重要性，以及当这种机制缺陷时在个体中发生的严重后果提供重要的见解。

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.