Analysis of BRCA1 Recombination Functions

BRCA1重组功能分析

• 批准号: 9045573
• 负责人: Ralph Scully
• 金额: $ 32.87万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9045573
• 关键词: African American; Alleles; BRCA1 Mutation; BRCA1 gene; BRCA2 gene; Bayesian Modeling; Benign; Biological Assay; Breast; Cells; Classification; Clinical Data; Complex; DNA; DNA Damage; DNA Restriction Enzymes; DNA replication fork; Defect; Development; Double Strand Break Repair; Escherichia coli; Event; Flow Cytometry; Frequencies; Gene Conversion; Generic Drugs; Genes; Genetic Recombination; Genomic Instability; Germ Lines; Goals; Health; Human; Individual; Inherited; International Agency for Research on Cancer; Length; Life; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Measures; Missense Mutation; Modeling; Mus; Mutation; Nuclear; Pathogenicity; Pathway interactions; Poly(ADP-ribose) Polymerases; Population; Probability; Problem Solving; Proteins; Reporter; Risk; Role; S Phase; Sister Chromatid; Site; Susceptibility Gene; System; Testing; Tumor Suppression; Tumor Suppressor Proteins; Variant; Woman; Work; breast tumorigenesis; cancer risk; genetic analysis; homologous recombination; inhibitor/antagonist; innovation; malignant breast neoplasm; mouse model; mutant; novel; prophylactic; red fluorescent protein; repaired; risk variant; tool; tumorigenesis; variant of unknown significance

 DESCRIPTION (provided by applicant): BRCA1 and BRCA2 have established functions in homologous recombination (HR), particularly in the repair of replication-associated DNA breaks by sister chromatid recombination (SCR). Many rare BRCA1 missense mutant alleles, termed "variants of uncertain significance" (VUS), are difficult to classify as pathogenic or benign, due o their scarcity in the human population. Therefore, for a woman who carries a germ line BRCA1 VUS allele, the risk of developing breast or ovarian cancer is unknown. A major goal of this proposal is to study a large panel of ~150 BRCA1 variants, including ~60 BRCA1 VUS alleles in HR/SCR, using highly innovative SCR reporters that are unique to the Scully lab. Our work will reveal fundamental mechanisms of action of BRCA1 in HR. Further, it will advance human health by helping to predict cancer risk for individuals who carry a BRCA1 VUS allele in the germ line and, hence, make informed decisions regarding prophylactic measures. AIM 1: Perform a genetic analysis of a large panel of BRCA1 variants in homologous recombination and LTGC suppression. We developed novel flow cytometric reporters of SCR, triggered by a site-specific chromosomal double strand break (DSB). We discovered that, in addition to its known function in "overall HR", BRCA1 also specifically suppresses aberrant "long tract" gene conversion (LTGC) between sister chromatids. Each of these defects is corrected by wild type human (h)BRCA1 but not by known pathogenic missense hBRCA1 alleles. In this Aim we will extend our analysis to include a large number of BRCA1 variants, including VUS alleles, with the goal of developing a robust predictor of the pathogenicity of specific BRCA1 VUS alleles. This Aim will include analysis of BRCA1 SCR functions in normal development and during breast tumorigenesis in a mouse model. AIM 2: Define mechanisms by which BRCA1 controls hr at Tus/ter-arrested replication forks. A long-standing hypothesis proposes that BRCA1 controls HR at stalled replication forks. This has been difficult to test, due to the dearth of tractable tools for stalling the mammalian replication fork at a defined chromosomal locus. We recently solved this problem by adapting the Escherichia coli Tus/ter replication fork arrest complex for use in mammalian cells. We discovered that BRCA1 controls HR at Tus/ter-stalled replication forks, but that HR in this context is regulated differently from HR induced by a "generic" restriction endonuclease-induced chromosomal DSB. Remarkably, loss of wtBRCA1 causes an increase in the absolute frequency of LTGC events at Tus/ter-stalled replication forks. In this proposal, we will analyze how BRCA1 controls HR at stalled forks and will attempt to identify the specific mechanisms by which BRCA1 suppresses LTGC at Tus/ter-stalled mammalian chromosomal replication forks.

 描述（由应用程序提供）：BRCA1和BRCA2在同源重组（HR）中建立了功能，特别是在修复与复制相关的DNA断裂（姐妹染色单体重组（SCR））方面。许多稀有的BRCA1错义突变等位基因称为“不确定意义的变体”（VUS），由于它们在人口中稀缺而难以归类为致病性或良性。因此，对于携带生物系BRCA1 VUS等位基因的女性而言，尚不清楚乳腺癌或卵巢癌的风险。该提案的主要目的是使用Scully Lab独有的高度创新性SCR报告者研究HR/SCR中〜60个BRCA1 VUS等位基因的大型面板。我们的工作将揭示BRCA1在人力资源中的基本作用机制。此外，它将通过帮助预测种系中携带BRCA1 VUS等位基因的个体的癌症风险，从而提高人类健康，从而对抗抗性测量做出明智的决定。 AIM 1：对同源重组和LTGC抑制中的大量BRCA1变体进行遗传分析。我们开发了SCR的新型流式细胞术记者，由特定位点特异性染色体双链（DSB）触发。我们发现，除了其在“整体HR”中的已知功能外，BRCA1还特别抑制了姐妹染色单体之间异常的“长道”基因转换（LTGC）。这些缺陷中的每一个都通过野生型人（H）BRCA1纠正，但未通过已知的致病错义HBRCA1等位基因纠正。在此目标中，我们将扩展分析，以包括大量的BRCA1变体，包括VUS等位基因，目的是开发特定BRCA1 VUS等位基因致病性的可靠预测指标。该目标将包括对小鼠模型中正常发育和乳腺肿瘤发生过程中BRCA1 SCR功能的分析。 AIM 2：定义BRCA1在TUS/TER Arrested Replication Forks上控制HR的机制。长期以来，BRCA1在停滞的复制叉中控制HR的长期假设建议。由于可在定义的染色体基因座处停滞哺乳动物复制叉的可拖动工具死亡，因此很难测试。最近，我们通过调整大肠杆菌/ter复制叉式捕获复合物来解决此问题，以用于哺乳动物细胞。我们发现BRCA1控制着TUS/TER恒星复制叉处的HR，但是在这种情况下，HR的调节与由“通用”限制性核酸内切酶诱导的染色体DSB诱导的HR不同。值得注意的是，WTBRCA1的丢失会导致TUS/TER恒星复制叉处LTGC事件的绝对频率的增加。在此提案中，我们将分析BRCA1如何控制失速的叉子的HR，并试图确定BRCA1在TUS/TER粘贴的哺乳动物染色体复制叉处抑制LTGC的特定机制。