Characterization of replication-mediated DNA damage in B lymphocytes

B 淋巴细胞复制介导的 DNA 损伤的表征

基本信息

批准号：
8764124
负责人：
Jacqueline Barlow
金额：
$ 16.2万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8764124
关键词：
Affect Antigen Receptors Antigens B-Cell Development B-Cell Lymphomas B-Lymphocytes Binding Proteins Biological Models Burkitt Lymphoma Cells ChIP-seq Characteristics Chromatin Chromosome Fragile Sites Complex Copy Number Polymorphism Coupled Cyclin A Cyclin D1 Cytogenetic Analysis DNA DNA Damage DNA Repair DNA Sequence Alteration DNA biosynthesis DNA lesion DNA repair protein Development Diagnosis Drug resistance Drug usage Embryo Enzymes Equilibrium Euchromatin Event Evolution Exhibits Follicular Lymphoma Frequencies Gene Expression Gene Rearrangement Genes Genetic Transcription Genome Genome Stability Genomic Instability Genomics Goals Heavy-Chain Immunoglobulins Human Hybrids IGH@ gene cluster Immunoglobulin Class Switching Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination Induced Mutation Investigation Lead Light Link Location Lymphocyte Lymphoma Lymphomagenesis MYC gene Malignant Neoplasms Maps Massive Parallel Sequencing Mediating Methods Molecular Monitor Mus Mutation Oncogenes Oncogenic Phosphotransferases Play Poison Precipitation Predisposing Factor Process Proteins RNA Receptor Gene Recovery Recurrence Regulation Replication Initiation Replication Origin Risk Role S Phase STAT6 gene Site Source Stimulus Stress Structure Techniques Testing Time activation-induced cytidine deaminase c-Myc Staining Method c-myc Genes cancer cell cancer genome cancer initiation cell type chromatin immunoprecipitation genome-wide helicase homologous recombination hydroxyurea improved insertion/deletion mutation novel null mutation overexpression paired box 5 protein (B-cell lineage specific activator)premature prevent programs public health relevance repaired research study response response marker senescence stem tool transcriptome sequencing tumor tumor progression tumorigenesis

项目摘要

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.

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 复制应激如何导致涉及癌症发生和进展的 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经历大规模增殖并响应抗原刺激，启动体细胞超突变 (SH) 和类别转换重组 (CSR) 的程序性 DNA 突变和重排过程。虽然负责产生 DNA 损伤并启动程序性 DNA 重排的酶已被证明在致癌易位中发挥着重要作用，但人们对复制应激如何促进这一过程却知之甚少。为了识别复制应激引起的新的脆弱位点，我们使用羟基脲 (HU) 药物绘制了早期 S 期发生的 DNA 损伤位点。通过使用染色质沉淀和大规模平行测序 (ChIP-Seq) 来监测单链结合蛋白 RPA、DNA 损伤反应标记 g-H2AX 以及同源重组 (HR) 蛋白 Brca1 和 Smc5 的协调募集，以响应 HU ，我们确定了被指定为早期复制脆弱位点（ERFS）的首选基因组位点，这些位点容易受到过早的分叉崩溃的影响。我们证实，这些位点确实对复制相关的 DNA 应激高度敏感，在检查点激酶 ATR 抑制或 c-Myc 癌基因过度表达时表现出 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 并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。这些结果导致了这样的假设：复制起始或转录活性的错误调节通过改变复制起始位点的频率和位置来诱导基因组不稳定。为了检验这一假设，我建议通过两种方式研究使特定基因组位点易受复制应激的分子因素：1）检查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。复制损伤的修复可能导致插入、缺失和复杂的重排事件，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二次突变，诱导促进适应和化疗耐药性的突变。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的进一步重排和进化的基本理解。