FUNCTIONAL ANALYSIS OF BRCA1 MUTANTS

BRCA1 突变体的功能分析

• 批准号: 6933003
• 负责人: Alvaro N Monteiro
• 金额: $ 23.23万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933003
• 关键词: DNA damage; DNA repair; alleles; brca gene; gamma radiation; gene mutation; genetic regulation; genetic transcription; histones; neoplasm /cancer genetics; phosphorylation; protein localization; temperature sensitive mutant; tissue /cell culture

Breast cancer is the most frequent malignancy affecting women in the United States. Hereditary breast and ovarian cancer represent 5-10 percent of all cases, most of which are attributed to inherited mutations in the tumor suppressor gene BRCA1. These mutations are highly penetrant and confer a 56-85 percent lifetime risk. The human BRCA1 gene encodes a protein product, which is likely to be multifunctional. The exact role of BRCA1 is still unclear but recent evidence points to the involvement of BRCA1 in two fundamental cellular processes: transcriptional regulation and DNA repair. To date, there is a wealth of evidence implicating BRCA1 in a variety of different DNA repair processes yet none of the evidence indicates a potential biological mechanism by which BRCA1 may act. These functions of BRCA1 may reflect two separate activities or, alternatively, part or all of the functions of BRCA1 in maintaining genome integrity may be an indirect result of BRCA1-mediated regulation of gene expression. Both scenarios are consistent with the current evidence. Our long-range goal is to test our central hypothesis that BRCA1 modulates transcription and that the modulation is important for efficient DNA repair. We have generated strong preliminary data for the role of BRCA1 in transcription. In addition, we have discovered a cancer-predisposing allele of BRCA1 that displays temperature-sensitive activity, providing a novel experimental system. Temperature-sensitive (TS) mutants constitute an invaluable tool to probe for the function of a particular protein. The objective of this application, which is one step towards our long-range goal, is to develop assays and characterize reagents that will be used to test whether transcriptional activation and control of DNA repair processes are interconnected functions of BRCA1. The rationale for the proposed research is that knowledge of the role of BRCA1 will allow us to design innovative approaches to counteract the effects of BRCA1 deficiency in cells and to develop functional assays for risk assessment. We will achieve our objective by accomplishing the following three specific aims: 1) Using our temperature-sensitive mutant of BRCA1, we will determine the time course of BRCA1 requirement for resistance to gamma-irradiation. 2) Establish whether subnuclear localization or hyperphosphorylation of TS BRCA1 are compromised at restrictive temperatures. 3) Determine the effects of DNA damage on transcription regulation by BRCA1. It is our expectation that the proposed research will ultimately determine the role of BRCA1 in the regulation of transcription and its connections to DNA damage response. These results will have considerable biomedical significance in identifying new targets for preventive and therapeutic intervention against women's cancers.

乳腺癌是影响美国女性的最常见的恶性肿瘤。 遗传性乳腺癌和卵巢癌占所有病例的 5-10%，其中大部分归因于肿瘤抑制基因 BRCA1 的遗传突变。 这些突变具有高度渗透性，终身风险高达 56-85%。 人类 BRCA1 基因编码一种蛋白质产物，该蛋白质产物可能具有多功能性。 BRCA1 的确切作用仍不清楚，但最近的证据表明 BRCA1 参与两个基本细胞过程：转录调节和 DNA 修复。 迄今为止，有大量证据表明 BRCA1 参与多种不同的 DNA 修复过程，但没有任何证据表明 BRCA1 可能发挥作用的潜在生物学机制。 BRCA1 的这些功能可能反映了两种独立的活性，或者，BRCA1 在维持基因组完整性方面的部分或全部功能可能是 BRCA1 介导的基因表达调节的间接结果。 这两种情况都与当前证据一致。 我们的长期目标是检验我们的中心假设，即 BRCA1 调节转录，并且这种调节对于有效的 DNA 修复很重要。 我们已经生成了关于 BRCA1 在转录中的作用的强有力的初步数据。 此外，我们还发现了 BRCA1 的一种致癌等位基因，它表现出温度敏感活性，从而提供了一种新颖的实验系统。 温度敏感（TS）突变体是探测特定蛋白质功能的宝贵工具。 该应用的目的是开发检测方法和表征试剂，用于测试转录激活和 DNA 修复过程的控制是否是 BRCA1 的相互关联的功能，这是实现我们长期目标的一步。 拟议研究的基本原理是，了解 BRCA1 的作用将使我们能够设计创新方法来抵消细胞中 BRCA1 缺陷的影响，并开发用于风险评估的功能测定。 我们将通过实现以下三个具体目标来实现我们的目标：1）使用我们的 BRCA1 温度敏感突变体，我们将确定 BRCA1 抵抗伽马射线照射所需的时间过程。 2) 确定 TS BRCA1 的亚核定位或过度磷酸化是否在限制温度下受到损害。 3) 确定DNA损伤对BRCA1转录调控的影响。 我们期望所提出的研究将最终确定 BRCA1 在转录调节中的作用及其与 DNA 损伤反应的联系。 这些结果对于确定女性癌症预防和治疗干预的新目标具有相当大的生物医学意义。