Genetic Dissection of BRCA1's Recombination Function

BRCA1重组功能的基因剖析

基本信息

批准号：
6897601
负责人：
DOUGLAS K BISHOP
金额：
$ 30.54万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
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/6897601
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

项目摘要

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 的功能，这些功能有助于其作为肿瘤抑制基因的作用。 80%的女性突变携带者一生中会患上乳腺癌。 BRCA1 和另一个主要乳腺癌易感基因 BRCA2 通过促进同源重组酶 RAD51 在受损位点的组装而参与 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。大量研究表明 BRCA1 与多种生物过程有关，表明该蛋白质具有多功能性。 BRCA1 是一种非常大的蛋白质，可直接或间接与许多蛋白质和 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研究，但人们对这些不同的相互作用如何与 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。蛋白质的哪些区域有助于特定功能？不同功能的功能之间是如何关联的？ BRCA1 的所有功能还是其中一部分功能都需要 DNA 结合？ BRCA1 通过什么机制有助于对阻止复制叉进展的药物产生耐药性？这些问题可以使用鸡前 B 细胞系 DT40 来解决，因为该细胞系可以通过分子遗传学轻松修改。该项目的目标如下。 1. 分析 DT40 的 BRCA1-/- null 突变体衍生物在同源重组、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 基因也是重组复合物组装所必需的。2. 通过表达谱寻找 BRCA1 调节转录物，并表征新发现的 BRCA1 调节基因的功能。 3. 开发一种新的检测方法来检查停滞复制叉的结构变化并确定 BRCA1 在这些变化中的作用。 4. 构建并表征一组细胞系，其中 BRCA1 区域被系统删除或改变，以绘制正常细胞环境中 BRCA1 的功能域。 5. 使用生化和遗传检测来绘制 BRCA1 有助于其 DNA 结合活性的区域，并确定 BRCA1 的哪些功能依赖于该活性。