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

DNA 链间交联 (ICL) 可以通过内源性（例如脂质过氧化）诱导 环境因素（例如吸烟、汽车尾气和污染）。 ICL 损坏系绳 DNA 双链体的两条链并阻断复制等重要的 DNA 代谢功能。它仍然存在 人们对 ICL 损伤如何在人体中修复知之甚少。 ICL 也是引起的主要毒性损伤 通过许多双功能化疗药物来杀死癌细胞。细胞对此类药物产生耐药性 通过上调 ICL 的修复能力，从而损害治疗效果。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、 癌症倾向、对交联剂过敏，表明 FA 蛋白参与 ICL 的修复和耐受性。迄今为止，至少已鉴定出 13 个 FANC 基因。八个（FANC-A、B、C、E、F、 G、L 和 M）中的 13 种 FANC 基因产物存在于蛋白质复合物中，称为 FA 核心 复杂。根据初步数据和之前的观察，我们假设 FANCM 参与 在ICL修复的切口步骤中，FA核心复合物参与切口的调节 核酸内切酶活性可实现 ICL 的精确有效切割。该提案的总体目标是 描述 ICL 两侧双切口损伤（脱钩）的机制并确定如何 FA 核心复合体有助于维持复制叉的稳定性，并有助于 ICL 在以下情况下脱钩： DNA 复制叉遇到 ICL。我们将表征 FANCM 的酶特性，确定 进行 ICL 切口的核酸内切酶，并测试它们是否相互协作 ICL 成功脱钩。通过采用 RNA 干扰和 cDNA 互补分析，我们将验证 通过监测 ICL 切口诱导的 DNA 产生，在人成纤维细胞中进行体外发现 双链断裂。我们将测试 FA 核心复合体的组成部分如何参与维持稳定性 复制叉和调节 ICL 切口核酸内切酶的活性。我们将净化所有成分 FA 核心复合体，评估其 DNA 损伤识别活性，分析其物理和功能 与切口核酸内切酶的相互作用，并描绘了损伤切口的调节机制 生化定义的体外系统。我们还将确定 FA 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体细胞中的核酸内切酶。 了解ICL识别和切开的机制不仅有助于整体 阐明了ICL修复过程，也为介入策略提供了新颖的依据。例如， 开发 ICL 修复抑制剂将导致未来化学增敏剂的转化研究 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.