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

范可尼贫血核心复合物在 DNA 链间交联切口中的作用

• 批准号: 8197847
• 负责人: Yanbin Zhang
• 金额: $ 38.02万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197847
• 关键词: Automobile Exhaust; Binding; Biological; Cancer Patient; Cancerous; Cells; Chemotherapy-Oncologic Procedure; Collaborations; Complementary DNA; Complex; Confocal Microscopy; DNA; DNA Damage; DNA Double Strand Break; DNA Interstrand Cross-Link Repair; DNA Interstrand Crosslinking; DNA lesion; DNA replication fork; Data; Defect; Deoxyribonuclease I; Development; Drug resistance; ERCC1 gene; Fanconi' s Anemia; Fibroblasts; Future; Genes; Genetic; Goals; Health; Hereditary Breast Carcinoma; Hereditary Disease; Human; Hypersensitivity; In Vitro; Interference Microscopy; Intervention; Lead; Lesion; Lipid Peroxidation; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Metabolic; Monitor; Pancytopenia; Pharmaceutical Preparations; Pollution; Positioning Attribute; Predisposition; Process; Production; Property; Proteins; RNA Interference; Recruitment Activity; Regulation; Research; Research Proposals; Resistance development; Role; SS DNA BP; Side; Single-Stranded DNA; Surgical incisions; Syndrome; System; Testing; Toxic effect; Translational Research; Treatment Efficacy; UV incision nuclease; base; cancer therapy; chemosensitizing agent; cigarette smoking; crosslink; design; endonuclease; environmental agent; improved; in vitro Assay; in vivo; inhibitor/antagonist; insight; killings; novel; protein complex; repaired; tool

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.

DNA链间交联（ICL）可以内源性诱导（如脂质过氧化）， 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，并阻断必要的DNA代谢功能，如复制。它仍然是 对ICL损伤如何在人体中修复知之甚少。ICL也是诱发的主要毒性病变 许多双功能化疗药物杀死癌细胞。细胞对这些试剂产生抗性 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，其特征在于骨髓衰竭，发育缺陷， 易患癌症，对交联剂过敏，表明FA蛋白参与了 ICL的修复和耐受性。迄今为止，已经鉴定出至少13个FANC基因。八个（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 复杂.根据初步数据和以前的观察，我们假设FANCM参与了 在ICL修复的切口步骤中，FA核心复合体参与切口的调节 用于精确和有效切割ICL的核酸内切酶活性。本提案的总体目标是 描述ICL损伤两侧双切口的机制（脱钩），并确定如何 FA核心复合体有助于保持复制分叉的稳定性，并有助于ICL脱钩， DNA复制叉遇到ICL。我们将表征FANCM的酶性质，鉴定 进行ICL切割的核酸内切酶，并测试它们是否彼此协作， 成功的ICL脱钩。通过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的体外发现 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ICL切割核酸内切酶的调节活动。我们将净化所有成分 的FA核心复合物，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。 生物化学定义的体外系统。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来调节人类细胞中的核酸内切酶。 了解ICL识别和切割的机制不仅有助于整体的 ICL修复过程的澄清，而且还为干预策略提供了新的基础。比如说， 开发ICL修复抑制剂将导致未来对化学增敏剂的转化研究， 克服了临床上观察到的癌症患者的耐药性。