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: 8024956
• 负责人: Yanbin Zhang
• 金额: $ 35.9万
• 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/8024956
• 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

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.

描述（由申请人提供）：DNA间交联（ICL）可以内源性（例如脂质过氧化）和环境剂（例如香烟吸烟，汽车排气和污染）诱导。 ICL损伤将DNA双链体的两条链和阻断必需的DNA代谢功能（例如复制）。它仍然不太了解人类如何修复ICL损害。 ICL也是许多双官能化疗药物诱导的主要毒性病变，以杀死癌细胞。细胞通过上调其ICL的修复能力，从而损害了治疗功效，从而对此类药物产生抗性。 Fanconi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，对交联药的超敏反应，表明FA蛋白参与了ICL的修复和耐受性。到目前为止，已经发现了至少13个幻想基因。在蛋白质复合物中发现了八个（FANC-A，B，C，E，F，G，L和M），被称为FA核心复合物。基于初步数据和先前的观察结果，我们假设FANCM参与了ICL修复的切口步骤，而FA核心复合物参与了切口内切核酸酶活动的调节，以精确有效切口ICL。该提案的总体目的是描述ICL损坏两侧双重切口的机制（解开），并确定FA核心复合物如何帮助维持复制叉的稳定性，并在DNA复制时遇到ICL时会解开ICL。我们将表征FANCM的酶促特性，确定执行ICL切口的核核酸酶，并测试它们是否相互合作以成功地解开。通过采用RNA干扰和cDNA互补分析，我们将通过监测ICL切口诱导的DNA双链断裂的产生来验证人成纤维细胞中的体外发现。我们将测试FA核心复合物的组成部分如何参与维持复制叉的稳定性以及调节ICL切口核酶的活动。我们将净化FA核心复合物的所有组件，评估其DNA损伤识别活性，介绍其与切口核酸内切酶的物理和功能相互作用，并在体外定义的体外系统中描述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确定FA核心复合物的募集并调节人类细胞中的核酶。了解ICL识别和切口的机制不仅有助于对ICL修复过程的总体澄清，而且还为介入策略提供了新的基础。例如，开发ICL修复的抑制剂将导致化学效应器的未来转化研究，以克服癌症患者临床观察到的耐药性。 公共卫生相关性：DNA间链交联维修的表征与人类健康直接相关，因为DNA交联剂广泛用于癌症化学疗法和缺乏链链交联修复构​​成了容易发生癌症的遗传综合征，例如fanconi贫血和遗传性乳腺癌，以及遗传性乳腺癌和遗传性乳腺癌和卵巢癌。我们的提案研究将为设计有效的策略提供有意义的见解，以增强化学治疗药物的癌症特异性毒性并改善对易癌症综合症的治疗。