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

描述（由申请人提供）：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）诱导。ICL损伤束缚DNA双链体的两条链，并阻断必要的DNA代谢功能，如复制。人们对ICL损伤如何在人类中修复仍然知之甚少。ICL也是许多双功能化疗药物杀伤癌细胞所诱导的主要毒性病变。细胞通过上调其ICL的修复能力而对此类药剂产生抗性，从而损害治疗功效。范可尼贫血（FA，FANC）是一种遗传性疾病，其特征是骨髓衰竭、发育缺陷、癌症易感性、对交联剂超敏反应，表明FA蛋白参与ICL的修复和耐受。迄今为止，已经鉴定出至少13个FANC基因。十三种FANC基因产物中的八种（FANC-A、B、C、E、F、G、L和M）存在于蛋白质复合物中，称为FA核心复合物。基于初步的数据和先前的观察，我们假设FANCM参与ICL修复的切割步骤，并且FA核心复合物参与切割核酸内切酶活性的调节以精确和有效地切割ICL。该提案的总体目标是描绘ICL损伤两侧的双切口（脱钩）的机制，并确定FA核心复合物如何帮助维持复制叉的稳定性，以及当DNA复制叉遇到ICL时如何有助于ICL脱钩。我们将表征FANCM的酶性质，鉴定进行ICL切割的核酸内切酶，并测试它们是否相互协作以成功地ICL脱钩。通过采用RNA干扰和cDNA互补分析，我们将通过监测ICL切割诱导的DNA双链断裂的产生来验证在人成纤维细胞中的体外发现。我们将测试FA核心复合物的组分如何参与维持复制叉的稳定性和调节ICL切口核酸内切酶的活性。我们将纯化FA核心复合物的所有组分，评估它们的DNA损伤识别活性，描述它们与切口核酸内切酶的物理和功能相互作用，并描述在生化定义的体外系统中损伤切口的调节机制。我们还将通过RNA干扰和共聚焦显微镜来确定FA核心复合物是否招募和调节人类细胞中的核酸内切酶。了解ICL的识别和切割机制，不仅有助于全面阐明ICL的修复过程，而且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未来对化学增敏剂的转化研究，以克服临床观察到的癌症患者的耐药性。 公共卫生相关性：DNA链间交联修复的表征直接关系到人类健康，因为DNA交联剂广泛用于癌症化疗，并且链间交联修复缺陷是易患癌症的遗传综合征如范可尼贫血和遗传性乳腺癌和卵巢癌的基础。我们的提案研究将提供有意义的见解，设计有效的策略，以增强化疗药物的癌症特异性毒性和改善癌症易感综合征的治疗。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The characterization of DNA interstrand cross-link repair is directly related to human health because DNA cross-linking agents are widely used in cancer chemotherapy and deficient interstrand cross-link repair underlies the cancer-prone genetic syndromes such as Fanconi anemia and hereditary breast and ovarian cancers. Our proposal research will provide meaningful insights to the design of effective strategies for potentiating the cancer-specific toxicity of chemotherapeutic drugs and improving therapy for the cancer-prone syndromes.