ISOLATION OF THE FANCONI ANEMIA NUCLEAR PROTEIN COMPLEX

范可尼贫血核蛋白复合物的分离

• 批准号: 6659774
• 负责人: GARY M KUPFER
• 金额: $ 36.87万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-10 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6659774
• 关键词: DNA binding protein; cell cycle; cell line; congenital aplastic anemia; gel mobility shift assay; gene complementation; mass spectrometry; nuclear matrix; protein localization; protein purification

Fanconi anemia (FA) is a genetic disease with defects in development and hematopoiesis and propensity to cancer, indicating a vital and basic cell biology process at work. The hallmark of FA is genomic instability, evidenced by gross chromosomal breakage and DNA alkylating agent hypersensitivity, which correlates strongly with cancer predilection in general. Studies of FA are important in several ways. First, FA biology is involved across a spectrum of scientific disciplines, including hematology, oncology, and development. Second, since the known FA proteins are found only in mammalian cells and have no previously described protein domain, their study will yield the description of a novel pathway which promotes the maintenance of gnomic stability. Third, work on other proteins derived from rare cancer susceptibility syndromes have proved to have wide applicability in science in general and cancer in particular, such as Li-Fraumeni syndrome (p53), xeroderma pigmentosum (DNA nucleotide excision repair), and ataxia telangiectasia (P13 kinase). Fourth, basic work on FA has already led to clinical use of reagents for diagnosis and genetic counseling, and gene therapy trials are currently underway for treatment of FA. This work focuses on FA protein interactions. Preliminary data have shown that two FA proteins FANCA (Fanconi anemia complementation group A) and FANCC interact in a 500 kD nuclear complex, an interaction that is absent in 7 of the 8 FA complementation groups. Within the nucleus the data reveal that these proteins localize to the DNA and nuclear matrix-containing fractions and have an appearance that parallels that of other nuclear matrix proteins. Evidence is also presented for inducible localization to chromatin and for direct DNA binding on gel shift assay. The efforts in this proposal will be driven by and will directly test two hypotheses: l) that the FA nuclear complex functions as a multimeric complex whose isolation will yield additional binding partners which will enable us to clone FA complementation group genes, and 2) that the FA proteins are associated with the nuclear matrix and with DNA in a cell cycle-regulated, complementation group-dependent, and drug-inducible fashion and interact with DNA either directly or indirectly. First, the FA protein complex will be characterized with respect to size and inducibility. Second, The complex will be isolated and analyzed by mass spectroscopy in order to identify novel and known associated proteins. Third, the subcellular localization of the FA proteins and their interaction with DNA will be investigated in a number of ways, including those which can lead to further new protein isolation. Identification of new proteins and elucidation of FA pathway mechanisms will help uncover a new realm of cancer biology and directly provide clinical applicability.

范可尼贫血（FA）是一种遗传性疾病，具有发育和造血缺陷以及癌症倾向，表明重要且基本的细胞生物学过程正在发挥作用。 FA 的标志是基因组不稳定，表现为染色体严重断裂和 DNA 烷化剂过敏，这与癌症的总体患病风险密切相关。 FA 研究在几个方面都很重要。首先，FA 生物学涉及一系列科学学科，包括血液学、肿瘤学和发育学。其次，由于已知的 FA 蛋白仅在哺乳动物细胞中发现，并且没有先前描述的蛋白结构域，因此他们的研究将产生一种促进维持基因组稳定性的新途径的描述。 第三，对源自罕见癌症易感综合征的其他蛋白质的研究已被证明在一般科学和特别是癌症中具有广泛的适用性，例如李-法美尼综合征 (p53)、着色性干皮病（DNA 核苷酸切除修复）和共济失调性毛细血管扩张症。 P13 激酶）。第四，FA的基础工作已经导致诊断和遗传咨询试剂的临床应用，目前正在进行FA治疗的基因治疗试验。这项工作的重点是 FA 蛋白相互作用。初步数据显示，两种 FA 蛋白 FANCA（范可尼贫血互补组 A）和 FANCC 在 500 kD 的核复合物中相互作用，这种相互作用在 8 个 FA 互补组中的 7 个中不存在。在细胞核内，数据显示这些蛋白质定位于含有 DNA 和核基质的部分，并且具有与其他核基质蛋白质相似的外观。还提供了可诱导定位至染色质以及在凝胶迁移测定中直接 DNA 结合的证据。该提案中的工作将由两个假设驱动，并将直接检验两个假设：l) FA 核复合物作为多聚体复合物发挥作用，其分离将产生额外的结合配偶体，这将使我们能够克隆 FA 互补组基因，以及 2) FA 蛋白以细胞周期调节、互补组依赖性和药物诱导的方式与核基质和 DNA 相关，并直接或间接与 DNA 相互作用。 首先，将表征 FA 蛋白复合物的大小和诱导能力。其次，将通过质谱分离和分析该复合物，以鉴定新型和已知的相关蛋白质。第三，FA蛋白的亚细胞定位及其与DNA的相互作用将通过多种方式进行研究，包括那些可以导致进一步分离新蛋白的方式。新蛋白质的鉴定和FA途径机制的阐明将有助于揭示癌症生物学的新领域并直接提供临床应用性。