Role of the BRCA1 Associated Protein BAP1 in DNA Repair

我们先前发现BRCA1相关蛋白-1(BAP1)是一种核定位的泛素羧基末端水解酶，提示脱泛素酶可能在BRCA1的功能中起作用。在BRCA1家族中定义的BRCA1无名指的突变取消了BAP1结合。BAP1和BRCA1在发育过程中在时间和空间上共表达。人类BAP1基因定位于染色体3p21.3，在人类肿瘤中发生缺失和点突变，表明它是一种肿瘤抑制基因。然而，到目前为止，BAP1如何在DNA修复、转录调控和重组的BRCA1通路中发挥作用还不清楚。由于BRCA1缺失细胞在氧化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。H2 26小细胞肺癌细胞系含有野生型BRCA1，但由于3p21.3 BAP1基因座的纯合缺失，BAP1为空。在初步数据中，我们显示BAP1缺失细胞具有与BRCA1缺失细胞相同的TCR缺陷。将野生型BAP1导入BAP1缺失细胞，可完全修复TCR缺陷。然而，BAP1的工程化点突变(Cys91Ser)或两个独立的人类肿瘤衍生点突变(Ala95Asp和G1y178Val)均可消除UCH酶，但均未能恢复TCR。值得注意的是，包含BAP1相关基因UCHL3的UCH酶结构域(在模型底物上显示野生型UCH活性)与BAP1的BRCA1相互作用区域融合的嵌合BAP1分子也未能重建TCR。因此，我们发现BAP1在BRCA1转录偶联修复途径中发挥作用。这是首次报道的UCH酶的功能，并证实了该家族中存在底物专一性。我们将通过以下目的来研究BAP1调节依赖BRCA1的TCRDNA修复途径的机制(S)。具体来说：1.对BAP-1进行全面的结构-功能分析，确定TCR的分子决定因素。2.明确BAP-1在DNA损伤时的UCH活性及其调控。2.寻找稳定在BAP-1互补细胞中的蛋白质。这些研究将在核定位泛素水解、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.