FUNCTIONAL ANALYSIS OF BRCA1 MUTANTS

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，可带来56%-85%的终生风险。人类BRCA1基因编码一种蛋白质产物，很可能是多功能的。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：转录调控和DNA修复。到目前为止，有大量的证据表明BRCA1参与了各种不同的DNA修复过程，但没有证据表明BRCA1可能通过一种潜在的生物学机制发挥作用。BRCA1的这些功能可能反映了两种不同的活动，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介导的基因表达调控的间接结果。这两种情况都与当前的证据一致。我们的长期目标是测试我们的中心假设，即BRCA1调节转录，并且这种调节对于有效的DNA修复是重要的。我们已经为BRCA1在转录中的作用产生了强有力的初步数据。此外，我们还发现了BRCA1的致癌等位基因，该等位基因对温度敏感，这为我们提供了一种新的实验系统。温度敏感(TS)突变体是探索特定蛋白质功能的宝贵工具。这项应用的目标是开发检测方法和表征试剂，用于测试DNA修复过程的转录激活和控制是否与BRCA1的相互关联功能，这是我们朝着长期目标迈出的一步。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BRCA1缺陷对细胞的影响，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)利用我们的温度敏感突变体BRCA1，我们将确定BRCA1对伽马辐射的抗性要求的时间进程。2)确定TS BRCA1的亚核定位或过度磷酸化是否在限制性温度下受到损害。3)确定DNA损伤对BRCA1转录调控的影响。我们期望这项拟议的研究将最终确定BRCA1在转录调控中的作用及其与DNA损伤反应的联系。这些结果将对确定预防和治疗女性癌症的新靶点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.