Characterization of replication-mediated DNA damage in B lymphocytes

描述(由申请人提供)：本申请的目标是以小鼠B淋巴细胞为模型系统，了解DNA复制应激对DNA重排的影响，DNA重排与癌症的发生和发展有关。淋巴细胞特别容易受到复制损伤，因为它们在整个发育过程中经历了大量的增殖，并对抗原刺激做出反应，启动了程序性DNA突变和重排过程，如体细胞过度突变(SH)和类开关重组(CSR)。虽然负责产生DNA损伤、启动程序性DNA重排的酶已被证明在致癌易位中发挥重要作用，但复制应激如何促进这一过程尚不清楚。为了识别由复制应激引起的新的脆性基因座，我们使用药物羟基脲(Hu)定位了发生在S早期的DNA损伤部位。通过利用染色质沉淀和大规模平行测序(CHIP-SEQ)监测单链结合蛋白RPA、DNA损伤反应标记g-H2AX以及同源重组(HR)蛋白BRCA1和SMC5对HU的响应，我们发现了对早熟崩溃易感的早期复制脆性位点(ERFS)。我们证实，这些位点确实对复制相关的DNA压力高度敏感，表现出增加的DNA断裂和重排，以回应检查点激酶ATR或c-Myc癌基因过度表达的抑制。重要的是，我们确定的ERF参与了在人类淋巴瘤中观察到的易位事件。进一步的鉴定表明，ERFS不是随机分布在整个基因组中，而是优先与转录活性簇相关联。这些结果导致了一种假说，即复制起始或转录活性的错误调节通过改变复制起始点的频率和位置而导致基因组不稳定。为了验证这一假设，我建议通过两种方式研究使特定基因组座位易于受到复制应激的分子因素：1)检测导致转录偶联复制应激的分子因素；2)表征癌基因过度表达如何诱导复制介导的DNA损伤。复制损伤的修复可以导致插入、缺失和复杂的重排事件，这让人想起在癌症中观察到的情况。此外，复制损伤还可能在癌细胞中产生继发性突变，诱导促进适应和化疗耐药的突变。这些研究将加强我们对复制诱导的DNA损伤如何促进肿瘤发展以及癌症基因组进一步重排和进化的基本理解。

DESCRIPTION (provided by applicant): The goal of this application is to understand how DNA replication stress contributes to DNA rearrangements involved in cancer initiation and progression using mouse B lymphocytes as a model system. Lymphocytes are particularly prone to replication damage as they undergo massive bursts of proliferation throughout development and in response to antigen stimulation, initiating the programmed DNA mutation and rearrangement processes of somatic hypermutation (SH) and class switch recombination (CSR). While the enzymes responsible for generating the DNA lesions initiating programmed DNA rearrangements have been shown to play an important role in oncogenic translocation, little is known how replication stress contributes to this process. To identify novel fragile loci arising from replication stress, we mapped sites of DNA damage occurring in early S phase using the drug hydroxyurea (HU). By monitoring the coordinated recruitment of the single strand binding protein RPA, the DNA damage response marker g-H2AX, and the homologous recombination (HR) proteins Brca1 and Smc5 using chromatin precipitation followed by massive parallel sequencing (ChIP-Seq) in response to HU, we identified preferred genomic loci designated as early replicating fragile sites (ERFSs) that are susceptible to premature fork collapse. We confirmed that these sites are indeed hypersensitive to replication-associated DNA stress, exhibiting increased DNA breaks and rearrangements in response to either inhibition of the checkpoint kinase ATR or c-Myc oncogene overexpression. Importantly, the ERFS we identified are involved in translocation events observed in human lymphomas. Further characterization showed that ERFSs are not distributed randomly throughout the genome, but associate preferentially with transcriptionally active clusters. These results have led to the hypothesis that mis- regulation of replication initiation or transcriptional activity induces genomc instability by altering the frequency and location of replication initiation sites. To test this hypothesis, I propose to study the molecular factors that predispose specific genomic loci to replication stress in two ways: 1) examine molecular factors contributing to transcription-coupled replication stress, and 2) characterize how oncogene overexpression induces replication-mediated DNA damage. Repair of replication damage can result in insertions, deletions and complex rearrangement events, reminiscent to those observed in cancer. Furthermore, replication damage may also produce secondary mutations in cancer cells, inducing mutations that promote adaptation and chemoresistance. These studies will enhance our fundamental understanding on how replication-induced DNA damage contributes to tumor development and further rearrangement and evolution of the cancer genome.