Characterization of replication-mediated DNA damage in B lymphocytes

描述（由申请人提供）：本申请的目的是使用小鼠B淋巴细胞作为模型系统，了解DNA复制应激如何促进参与癌症起始和进展的DNA重排。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和响应于抗原刺激而经历大规模的增殖爆发，启动程序性DNA突变和体细胞超突变（SH）和类别转换重组（CSR）的重排过程。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，但对复制应激如何促成这一过程知之甚少。为了确定新的脆性基因座所产生的复制压力，我们映射的DNA损伤发生在早期S期使用药物的羟基脲（HU）的网站。通过监测单链结合蛋白RPA、DNA损伤反应标记物g-H2 AX和同源重组（HR）蛋白Brca 1和Smc 5的协调募集，使用染色质沉淀，然后进行大规模平行测序（ChIP-Seq）以响应HU，我们确定了指定为早期复制脆性位点（ERFS）的首选基因组位点，这些位点易受过早分叉崩溃的影响。我们证实这些位点确实对复制相关的DNA应激高度敏感，表现出对检查点激酶ATR或c-Myc癌基因过表达的抑制的DNA断裂和重排增加。重要的是，我们发现的ERFS参与了在人类淋巴瘤中观察到的易位事件。进一步的表征表明，ERFS不是随机分布在整个基因组，但优先与转录活性簇。这些结果导致了复制起始或转录活性的失调通过改变复制起始位点的频率和位置而诱导基因组不稳定性的假设。为了验证这一假设，我建议从两个方面研究使特定基因组位点易受复制应激影响的分子因素：1）研究有助于转录偶联复制应激的分子因素，2）表征癌基因过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那些。此外，复制损伤还可能在癌细胞中产生继发性突变，诱导促进适应和化学抗性的突变。这些研究将增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.