Role of Fanconi Anemia Core Complex in the Incision of DNA Interstrand Crosslinks

DNA链间交联(ICL)可由内源性(如脂质过氧化)和 环境因素(如吸烟、汽车尾气和污染)。ICL损坏系绳 DNA的两条链都是双链，并阻止复制等基本的DNA代谢功能。它仍然存在 人们对ICL损伤是如何在人类身上修复的知之甚少。ICL也是引起的主要毒性损害 通过多种双功能化疗药物杀灭癌细胞。细胞对这类药物产生抗药性 通过上调ICL的修复能力，从而影响治疗效果。范科尼 贫血(FA，FANC)是一种遗传性疾病，其特征是骨髓衰竭、发育缺陷、 对癌症的易感性，对交联剂的超敏，表明FA蛋白参与了 ICL的修复和耐受性。到目前为止，至少已经鉴定出13个FANC基因。八个(FANC-A、B、C、E、F、 13个FANC基因产物中的G、L和M)存在于称为FA核心的蛋白质复合体中 很复杂。根据初步数据和先前的观察，我们假设FANCM参与了 在ICL修复的切开步骤中，FA核心复合体参与了切开的调节 精确有效切开ICL的核酸内切酶活性。这项提案的总体目标是 描述双侧ICL损伤(脱钩)的机制，并确定如何 FA核心复合体有助于维持复制分叉的稳定性，并有助于ICL在以下情况下解钩 DNA复制叉遇到ICL。我们将对FANCM的酶性质进行表征，鉴定 进行ICL切开的内切酶，并测试它们是否相互协作 ICL解钩成功。通过使用RNA干扰和cDNA互补分析，我们将验证 通过监测ICL切开诱导DNA的产生在体外发现人成纤维细胞 双链断裂。我们将测试FA核心复合体的组件如何参与维护稳定 复制叉和调节ICL切割内切酶的活性。我们将提纯所有组件 评估其DNA损伤识别活性，描述其物理和功能 与切口酶的相互作用，并描绘了损伤伤口的调节机制。 生物化学定义的体外系统。我们还将确定英足总核心综合体是否招募和 通过RNA干扰和共聚焦显微镜调节人类细胞中的内切酶。 了解ICL识别和切割的机制不仅有助于从整体上 阐明ICL的修复过程，也为介入治疗策略提供了新的依据。例如, 开发ICL修复的抑制剂将导致化学增敏剂未来的翻译研究 克服临床观察到的癌症患者的耐药性。

DNA interstrand crosslinks (ICLs) can be induced endogenously (e.g. lipid peroxidation) and by environmental agents (e.g. cigarette smoking, automobile exhausts, and pollution). The ICL damage tethers both strands of DNA duplex and blocks essential DNA metabolic functions such as replication. It remains poorly understood how the ICL damage is repaired in humans. ICLs are also the primary toxic lesions induced by many bi-functional chemotherapeutic drugs to kill cancerous cells. Cells develop resistance to such agents through up-regulating their repair capacity of ICLs, thereby compromising the therapeutic efficacy. Fanconi anemia (FA, FANC) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancers, hypersensitivity to crosslinking agents, indicating involvement of FA proteins in repair and tolerance of ICLs. At least 13 FANC genes have been identified thus far. Eight (FANC-A, B, C, E, F, G, L, and M) of the thirteen FANC gene products are found in a protein complex, termed as the FA core complex. Based on the preliminary data and previous observations, we hypothesize that FANCM participates in the incision step of ICL repair, and the FA core complex is involved in the regulation of incision endonuclease activities for precise and efficient incision of ICLs. The overall goal of this proposal is to delineate the mechanism of the dual incisions on both sides of ICL damage (unhooking) and to determine how the FA core complex helps maintaining stability of replication forks and contributes to the ICL unhooking when the DNA replication fork encounters an ICL. We will characterize the enzymatic properties of FANCM, identify the endonucleases that carry out the ICL incision, and test whether they collaborate with each other for successful ICL unhooking. By employing RNA interference and cDNA complementation analyses, we will verify the in vitro discoveries in human fibroblast cells through monitoring the ICL incision-induced production of DNA double strand breaks. We will test how components of the FA core complex are involved in maintaining stability of replication forks and in regulating activities of the ICL incision endonucleases. We will purify all components of the FA core complex, evaluate their DNA damage recognition activity, profile their physical and functional interactions with the incision endonucleases, and delineate the regulatory mechanism of damage incision in a biochemically defined in vitro system. We will also determine whether the FA core complex recruits and regulates the endonucleases in human cells through RNA interference and confocal microscopy. Understanding the mechanism of the ICL recognition and incision will not only contribute to the overall clarification of the ICL repair process, but also provide a novel basis for interventional strategies. For example, developing inhibitors of the ICL repair would lead to future translational research for chemosensitizers to overcome the clinically observed drug resistance in cancer patients.