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）：本应用的目的是了解DNA复制应力如何促进使用小鼠B淋巴细胞作为模型系统参与癌症开始和进展的DNA重排。淋巴细胞特别容易出现复制损伤，因为它们在整个发育过程中都会发生大规模的增殖，并响应抗原刺激，启动了躯体超突变（SH）和类转换重组（CSR）的编程的DNA突变和重排过程。虽然已经显示出启动编程的DNA重排的DNA病变的酶在致癌易位中起着重要作用，但鲜为人知的复制应力如何对这一过程产生影响。为了确定由复制应力引起的新型脆弱基因座，我们使用药物羟基脲（HU）绘制了在早期S期发生的DNA损伤部位。 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叉子过早倒塌。我们证实，这些位点确实对复制相关的DNA胁迫过敏，这表现出增加的DNA断裂和重排，以应对抑制检查点激酶ATR或C-Myc癌基因过表达。重要的是，我们确定的ERF参与了在人淋巴瘤中观察到的易位事件。进一步的表征表明，ERFS不是在整个基因组中随机分布的，而是与转录活性簇优先分布。这些结果导致了以下假设：误导复制起始或转录活性通过改变复制起始位点的频率和位置来引起基因组不稳定。为了检验这一假设，我建议研究以两种方式倾向于特异性基因组基因局基因组基因局重复应力的分子因子：1）检查有助于转录偶联复制应力的分子因素，以及2）表征癌基因过度表达如何诱导复制介导的介导的DNA损害。修复复制损伤可能会导致插入，缺失和复杂的重排事件，让人联想到癌症中观察到的事件。此外，复制损伤还可能在癌细胞中产生次要突变，从而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损伤如何有助于肿瘤发展以及癌症基因组进一步重排和进化的基本理解。