The molecular mechanism of DNA interstrand crosslink repair in humans

描述(申请人提供)：DNA链间交联(ICL)是一种细胞毒性DNA损伤，共价连接两条DNA链，阻止DNA复制和RNA转录。ICL是几种最常用的化疗药物引起的原发病变，包括顺铂和双功能烷化剂。因此，这些药物的疗效取决于修复ICL的效率，以及对细胞如何修复ICL的更好理解可能导致化疗药物和/或方案的改进。然而，人类细胞中ICL修复的分子机制(S)仍然知之甚少。本文提出的研究将利用一种新的体外ICL修复方法来研究人细胞ICL修复的分子机制(S)。该方法使用具有单一补骨脂素ICL和SV40复制起始点的特定底物DNA。初步结果表明，人类蛋白质对该底物中ICL的修复与DNA复制有关，需要BRCA2。先前的研究表明，人类细胞的ICL修复是通过以下四个动力学上不同的步骤完成的：(1)ICL在一条链上的解钩和诱导依赖DNA复制的双链断裂(DSB)，(2)跨损伤DNA合成(TLS)跨越去挂钩的ICL，(3)处理DSB和修复停滞的DNA复制叉，以及(4)去除残留的未连接的ICL。该方案的目的是确定完成ICL修复所需的人类蛋白质因子，并利用体外ICL修复实验确定这些蛋白质在ICL修复中所起的作用。该建议的具体目的是：1)确定结构特异性内切酶复合体XPF/ERCC1和MUS81/MMS4在人ICL修复中的作用；2)定义TLS聚合酶POLQ和PolN在使用纯化的His标记的人蛋白和带有未连接补骨脂素ICL的缺口DNA底物进行ICL修复中的作用；3)定义DSB修复蛋白在ICL修复中的作用，尤其是BRCA2和Fanconi贫血(FA)蛋白，它们的失活使人对DNA交联剂超敏。这里提出的实验将使用RNAi来分析ICL修复的每一步所需的蛋白质。ICL修复试验将与RNAi处理的人类细胞的提取物进行，并将通过分析ICL修复反应中积累的反应产物或中间体的结构和数量来推断被击倒的蛋白的作用。被击倒的提取物的结果也将通过重新加入纯化的蛋白质来确认。这些研究将有助于我们对人ICL修复的基本了解，并为利用纯化的修复蛋白在体外重建ICL修复奠定基础。公共卫生相关性：DNA链间交联(ICL)修复缺陷导致人类细胞遗传不稳定，并导致罕见的遗传性疾病，如家族性乳腺癌和Fanconi贫血(FA)。本文提出的这项研究将鉴定和表征与使用一种新的无细胞ICL修复试验有关的人类蛋白质组分。这些研究结果将有助于我们更好地理解ICL修复的分子机制。此外，拟议的实验还将研究BRCA2的结构-功能关系，并破译FA蛋白在ICL修复中的功能。这些研究将为理解BRCA2和FA蛋白的肿瘤抑制功能提供新的见解。

DESCRIPTION (provided by applicant): DNA interstrand crosslinks (ICLs) are cytotoxic DNA lesions that covalently link the two strands of DNA and block DNA replication and RNA transcription. ICLs are the primary lesion generated by several of the most commonly used chemotherapeutic agents, including cisplatin and bi-functional alkylating agents. Thus, the efficacy of these agents depends on the efficiency with which ICLs are repaired, and better understanding of how cells repair ICLs may lead to improved chemotherapeutic agents and/or protocols. Nevertheless, the molecular mechanism(s) of ICL repair in human cells remains poorly understood. The research proposed here will study the molecular mechanism(s) of ICL repair in human cells using a novel in vitro assay for ICL repair. This assay uses a defined substrate DNA with a single psoralen ICL and the SV40 origin of replication. Preliminary results indicate that repair of the ICL in this substrate by human proteins is coupled to DNA replication, and requires BRCA2. Previous studies suggest that ICL repair in human cells is accomplished in the following four kinetically distinct steps: (1) unhooking of the ICL on one strand and induction of a DNA replication dependent double-strand break (DSB), (2) translesion DNA synthesis (TLS) across from the unhooked ICL, (3) processing of the DSB and restoration of the stalled DNA replication fork, and (4) removal of the residual unhooked ICL. The goal of this proposal is to define the human protein factors required to accomplish ICL repair and to define the roles played by these proteins in ICL repair using the in vitro ICL repair assay. The specific aims of the proposal are: 1) Define the role of structure-specific endonuclease complexes XPF/ERCC1 and MUS81/MMS4 in human ICL repair; 2) Define the roles of TLS polymerases PolQ and PolN in ICL repair using purified His-tagged human proteins and a gapped DNA substrate with an unhooked psoralen ICL; 3) Define the role of DSB repair proteins in ICL repair with particular focus on BRCA2 and Fanconi anemia (FA) proteins, whose inactivation confers hypersensitivity to DNA crosslinking agents. The experiments proposed here will use RNAi to analyze the proteins required for each step of ICL repair. ICL repair assays will be carried out with extracts from RNAi-treated human cells, and the role of the knocked-down protein will be deduced by analyzing the structure and amount of the reaction products or intermediates that accumulate in the ICL repair reaction. Results with knocked down extracts will also be confirmed by adding back purified proteins. These studies will contribute to our basic understanding of human ICL repair and lay the groundwork for reconstituting ICL repair in vitro using purified repair proteins. PUBLIC HEALTH RELEVANCE: Defects in DNA interstrand crosslink (ICL) repair lead to genetic instability in human cells and cause rare genetic diseases such as familial breast cancer and Fanconi anemia (FA). This research proposed here will identify and characterize human protein components involved in using a novel cell-free ICL repair assay. The results of the proposed studies will improve our understanding of the molecular mechanism of ICL repair in human cells. In addition, the proposed experiments will also investigate structure-function relationships in BRCA2 and decipher the function of FA proteins in ICL repair. These studies will give new insights into understanding tumor suppressor functions of BRCA2 and FA proteins.