MOLECULAR MECHANISMS OF BRCA1-DEPENDENT DNA DAMAGE RESPONSE AND TUMORIGENESIS

描述（申请人提供）：BRCA1是一种抑制家族性乳腺癌和卵巢癌的核多肽。越来越多的证据表明BRCA1参与DNA损伤反应。然而，BRCA1参与DNA损伤反应的分子机制尚不清楚。最近，我们发现了两个新的BRCA1合作伙伴，RAP80和CCDC98。RAP80和CCDC98都与BRCA1 BRCT结构域相关，参与DNA损伤应答。功能上，RAP80和CCDC98促进BRCA1易位到DNA损伤位点。为了寻找将BRCA1复合体招募到DNA损伤病灶的信号，我们发现BRCA1相关蛋白RAP80识别泛素化组蛋白H2A和H2B。组蛋白H2A和H2B在DNA损伤后进一步泛素化。此外，我们在乳腺癌和卵巢癌细胞中发现了两个双等位基因错义突变和一个RAP80基因截断突变，提示RAP80可能是brca1依赖通路中另一个乳腺和卵巢肿瘤抑制因子。因此，我们假设RAP80对泛素化组蛋白的识别是将BRCA1装载到DNA损伤位点的分子基础，从而调节适当的DNA损伤反应，保护基因组完整性，防止乳腺和卵巢肿瘤的发生。我们提出以下实验来检验我们的假设。

DESCRIPTION (provided by applicant): BRCA1 is a nuclear polypeptide to suppress familial breast and ovarian cancers. Accumulated evidence suggests that BRCA1 participates in DNA damage response. However, the molecular mechanisms by which BRCA1 participates in DNA damage response remain elusive. Recently, we have identified two new BRCA1 partners, RAP80 and CCDC98. Both RAP80 and CCDC98 associate with BRCA1 BRCT domain and participate in DNA damage response. Functionally, RAP80 and CCDC98 facilitate BRCA1's translocation to DNA damage sites. To search for the signals that recruit this BRCA1 complex to the DNA damage lesions, we have found that BRCA1-associated protein RAP80 recognizes ubiquitinated histone H2A and H2B. And both histone H2A and H2B are further ubiquitinated following DNA damage. In addition, we have identified two biallelic missense mutations and one truncation mutation of RAP80 gene in breast and ovarian cancer cells, suggesting that RAP80 could be another breast and ovarian tumors suppressor in BRCA1-dependent pathway. Thus, we hypothesize that recognition of ubiquitinated histone by RAP80 is the molecular basis that loads BRCA1 to DNA damage sites, which regulates proper DNA damage response, protects genomic integrity and prevents breast and ovarian tumor development. We propose following experiments to examine our hypothesis. Aim1: To examine the molecular mechanism by which RAP80 and CCDC98 target BRCA1 to DNA damage lesions. Aim2: To examine the functional defects of RAP80 mutations in BRCA1-dependent DNA damage response. Aim3: To examine the role of RAP80 in tumor prevention. In summary, studies outlined here will not only reveal the molecular mechanism by which BRCA1 participates in DNA damage response, but also identify the functional partners of BRCA1 in tumor suppression. PUBLIC HEALTH RELEVANCE: Accumulated evidence suggests that BRCA1 protects genomic integrity in response to DNA double strand breaks. Here, we have identified two BRCA1 partners, CCDC98 and RAP80, which participate in DNA damage response. In this proposal, we plan to not only dissect the molecular mechanism of this BRCA1 complex in DNA damage response, but also examine the role of this complex in tumorigenesis in vivo.