Genetic Dissection of BRCA1's Recombination Function

BRCA1重组功能的基因剖析

• 批准号: 7059384
• 负责人: DOUGLAS K BISHOP
• 金额: $ 29.82万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059384
• 关键词: DNA binding protein; DNA damage; DNA replication origin; brca gene; cell cycle; cell line; cytogenetics; gene expression; genetic recombination; genetic transcription; neoplasm /cancer genetics; northern blottings; polymerase chain reaction; protein structure function; technology /technique development; tumor suppressor proteins

DESCRIPTION (provided by applicant): Our goal is to understand the functions of BRCA1 that contribute to its role as a tumor suppressor gene. 80% of female mutation carriers will develop breast cancer in their lifetime. BRCA1 and the other major breast cancer susceptibility gene BRCA2 have been implicated in recombinational repair of DNA damage by promoting the assembly of the homologous recombinase RAD51 at damaged sites. BRCA1's function does not appear to be limited to promoting recombinational repair, however. A large amount of work has implicated BRCA1 in diverse biological processes suggesting that the protein is multifunctional. BRCA1 is a very large protein and interacts, directly or indirectly, with many proteins and with DNA. These different protein-protein and proten-DNA interactions have been mapped to different regions of BRCA1 suggesting a structure composed of functional modules. In spite of the large amount of work on the protein, there is relatively little known about how these different interactions relate to BRCA1's different cellular functions in recombinational repair, base-excision repair, cell cycle checkpoint control, and gene expression. Which regions of the protein contribute to a given function? How are the different functions of the functions related? Is DNA binding required for all BRCA1's functions or just a subset? Through what mechanism does BRCA1 contribute to resistance to agents that block replication fork progression? These questions are addressed using the chicken pre-B cell line DT40 because of the ease with which this line can be modified by molecular genetics. The aims of the project are as follows. 1. To analyze the phenotypes of a BRCA1-/- null mutant derivative of DT40 with respect to homologous recombination, sensitivity to DNA damage, cell cycle checkpoint control, and transcription regulation. These phenotypes will be compared to those conferred by mutation of the XRCC3 gene, which is also required for assembly of recombination complexes 2. To search for BRCA1 regulated transcripts by expression profiling and to characterize the function of a newly discovered BRCA1 regulated gene. 3. To develop a new assay for examining structural changes of stalled replication forks and determine BRCA1's role in these changes. 4. To construct and characterize a set of cell lines in which regions of BRCA1 are systematically deleted or altered to map functional domains of BRCA1 in the normal cellular context. 5. To use biochemical and genetic assays to map the regions of BRCA1 that contribute to its DNA binding activity and determine which functions of BRCA1 depend on this activity.

描述（由申请人提供）：我们的目标是了解BRCA1作为肿瘤抑制基因的功能。80%的女性突变携带者会在一生中患上乳腺癌。BRCA1和其他主要的乳腺癌易感基因BRCA2通过促进同源重组酶RAD51在受损部位的组装而参与DNA损伤的重组修复。然而，BRCA1的功能似乎并不局限于促进重组修复。大量的研究表明BRCA1参与多种生物过程，这表明该蛋白是多功能的。BRCA1是一种非常大的蛋白质，直接或间接地与许多蛋白质和DNA相互作用。这些不同的蛋白质-蛋白质和蛋白质- dna相互作用已被映射到BRCA1的不同区域，表明其结构由功能模块组成。尽管对该蛋白进行了大量的研究，但相对而言，人们对这些不同的相互作用与BRCA1在重组修复、碱基切除修复、细胞周期检查点控制和基因表达中的不同细胞功能之间的关系知之甚少。蛋白质的哪些区域对给定的功能有贡献？函数的不同功能是如何联系在一起的？DNA结合是BRCA1所有功能所必需的还是仅仅是一个子集？BRCA1通过什么机制促进对阻断复制叉进展的药物的抗性？这些问题都是用鸡前b细胞系DT40来解决的，因为这种细胞系可以通过分子遗传学进行修饰。该项目的目标如下。1. 分析DT40的BRCA1-/- null突变衍生物在同源重组、对DNA损伤的敏感性、细胞周期检查点控制和转录调控方面的表型。这些表型将与XRCC3基因突变所赋予的表型进行比较，XRCC3基因突变也是重组复合物组装所必需的2。通过表达谱寻找BRCA1调控转录本，并对新发现的BRCA1调控基因的功能进行表征。3. 开发一种新的检测方法来检测停滞复制分叉的结构变化，并确定BRCA1在这些变化中的作用。4. 构建和表征一组细胞系，在这些细胞系中，BRCA1区域被系统地删除或改变，以绘制正常细胞环境下BRCA1的功能域。5. 利用生物化学和遗传分析绘制BRCA1中与DNA结合活性有关的区域，并确定BRCA1的哪些功能依赖于这种活性。