FUNCTIONAL ANALYSIS OF BRCA1 MUTANTS

乳腺癌是影响美国妇女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%，其中大多数归因于肿瘤抑制基因BRCA1的遗传突变。这些突变具有很高的渗透性，并具有56% - 85%的终生风险。人类BRCA1基因编码一种蛋白质产物，它可能是多功能的。BRCA1的确切作用尚不清楚，但最近的证据表明，BRCA1参与了两个基本的细胞过程：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动，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。这两种情况都与目前的证据一致。我们的长期目标是验证我们的中心假设，即BRCA1调节转录，并且这种调节对有效的DNA修复很重要。我们已经获得了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的一个易患癌症的等位基因，它显示出温度敏感的活性，提供了一个新的实验系统。温度敏感（TS）突变体是探测特定蛋白质功能的宝贵工具。该应用程序的目的是开发用于测试转录激活和DNA修复过程控制是否与BRCA1相互关联功能的检测方法和表征试剂，这是我们实现长期目标的一步。这项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的方法来抵消细胞中BRCA1缺陷的影响，并开发用于风险评估的功能分析。我们将通过实现以下三个具体目标来实现我们的目标：1)利用我们的BRCA1温度敏感突变体，我们将确定BRCA1抵抗γ辐照所需的时间过程。2)确定在限制性温度下，TS BRCA1的亚核定位或过磷酸化是否受到损害。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在转录调控中的作用及其与DNA损伤反应的联系。这些结果将在确定针对妇女癌症的预防和治疗干预的新目标方面具有相当大的生物医学意义。

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.