Role of the BRCA1 Associated Protein BAP1 in DNA Repair

我们之前在筛选BRCA1环指结构域结合蛋白的过程中发现了BRCA1相关蛋白-1 (BAP1)， BAP1是一种核定位的泛素羧基末端水解酶，这表明去泛素化酶可能在BRCA1的功能中发挥作用。在BRCA1家族中定义的BRCA1无名指突变会破坏BAP1的结合。在发育过程中，BAP1和BRCA1在时间和空间上共同表达。人类BAP1基因定位于染色体3p21.3，在人类肿瘤中存在缺失和点突变，是一种肿瘤抑制因子。然而，到目前为止，人们还不清楚BAP1如何在提出的BRCA1 DNA修复、转录调控和重组途径中发挥作用。由于brca1缺失的细胞在氧化DNA损伤的转录偶联修复（TCR）中存在缺陷，我们研究了BAP1在TCR中的作用。H226小细胞肺癌细胞系含有野生型BRCA1，但由于3p21.3 BAP1位点的纯合缺失，BAP1为零。在初步数据中，我们发现BAP1-null细胞具有与BRCA1-null细胞相同的TCR缺陷。将野生型BAP1转染到无BAP1的细胞中，完全恢复了TCR缺陷。然而，转染BAP1的工程点突变体（Cys91Ser）或两个独立的人类肿瘤来源的点突变体（Ala95Asp和G1y178Val），它们都能消除UCH酶，但都不能恢复TCR。值得注意的是，含有BAP1相关基因UCHL3的UCH酶结构域的嵌合BAP1分子（在模型底物上显示野生型UCH活性）融合到BAP1的BRCA1相互作用区域也未能重建TCR。因此，我们发现BAP1在BRCA1转录偶联修复通路中起作用。这是首次报道的UCH酶的功能，并确定了该家族中存在底物特异性。我们将通过执行以下目标来解决BAP1调节brca1依赖性TCR DNA修复途径的机制。具体:1。对BAP-1进行全面的结构-功能分析，并确定TCR的分子决定因素。2. 定义UCH活性和BAP-1在DNA损伤反应中的调节。2. 寻找在BAP-1互补细胞中稳定的蛋白质。这些研究将在核定位的泛素水解、DNA修复过程和肿瘤抑制功能之间建立新的联系。

We have previously discovered the BRCA1-associated Protein-1 (BAP1) which was cloned in a screen for proteins which bind to the RING finger domain of BRCA1 BAP1 is a nuclear-localized, ubiquitin carboxyl- terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. Mutations in the BRCA1 RING finger defined in BRCA1 kindreds abolish BAP1 binding. BAP1 and BRCA1 are temporally and spatially co-expressed during development. The human BAP1 gene maps to chromosome 3p21.3 and suffers deletions and point mutations in human tumors that it is a tumor suppressor. However, until now, it has been unclear how BAP1 functioned in the proposed BRCA1 pathways of DNA repair, transcriptional regulation and recombination. Since BRCA1-null cells have a defect in the transcription-coupled repair (TCR) of oxidative DNA damage, we examined the role of BAP1 in TCR. The H226 small cell lung cancer cell line contains wild-type BRCA1 but is null for BAP1 due to a homozygous deletion at the 3p21.3 BAP1 locus. In preliminary data we show that BAP1-null cells have a TCR defect identical to BRCA1-null cells. Transfection of wild-type BAP1 into the BAP1-null cells completely restored the TCR defect. However, transfection of an engineered point mutant of BAP1 (Cys91Ser) or two independent human tumor-derived point mutations (Ala95Asp and G1y178Val) each of which abolishes the UCH enzymatic failed to restore TCR. Remarkably, a chimeric BAP1 molecule containing the UCH enzymatic domain of the BAP1-related gene UCHL3 (which shows wild-type UCH activity on model substrates) fused to the BRCA1 interaction region of BAP1 also failed to reconstitute TCR. Thus, we have discovered that BAP1 functions in the BRCA1 transcription-coupled repair pathway. This is the first reported function for a UCH enzyme and establishes that there is a substrate specificity among this family. We will address the mechanism(s) by which BAP1 regulates the BRCA1-dependent TCR DNA repair pathway by performing the following aims. Specifically: 1. Perform a comprehensive structure-function analysis of BAP-1 and define the molecular determinants for TCR. 2. Define the UCH activities and regulation of BAP-1 in response to DNA damage. 2. Search for proteins which are stabilized in BAP-1 complemented cells. These studies will establish a new link between nuclear-localized ubiquitin hydrolysis, DNA repair processes and tumor suppressor function.