Transcription, Chromatin and DNA repair

转录、染色质和 DNA 修复

基本信息

批准号：
7592472
负责人：
rafael c casellas
金额：
$ 87.09万
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7592472
关键词：
ATM Signaling Pathway ATM deficient Animals Antigens B-Cell Development B-Lymphocytes Biochemical Biological Assay Cell Cycle Cell Surface Receptors Cells Chromatin Chromosomal translocation Complex Confocal Microscopy DNA DNA Damage DNA Polymerase I DNA Repair DNA Repair Enzymes DNA lesion DNA repair protein DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase Data Enzymes Etoposide Event Gene Expression Genes Genetic Genetic Recombination Genetic Transcription Genotoxic Stress Goals Green Fluorescent Proteins Holoenzymes Human Immune system Immunoglobulin Class Switching Immunoglobulin Genes Immunoglobulin Somatic Hypermutation Immunoglobulin Switch Recombination Immunoglobulins Infection Kinetics Label Lasers Lead Life Lymphocyte Lymphocyte Function Malignant Neoplasms Mammals Manuscripts Measures Mediating Modeling Modification Monitor Mus Nature Numbers Pathway interactions Phase Photobleaching Polymerase Process Publishing RNA Polymerase I RNA Polymerase II RNA chemical synthesis Reaction Receptor Gene Recombinant DNA Regulation Reporter Reporting Ribosomal DNA Ribosomal RNA Role Run-On Assays Site System T-Cell Lymphoma T-Cell Receptor T-Lymphocyte Technology Time Transcription-Coupled Repair Ubiquitination V(D)J Recombination chromatin remodeling irradiation mathematical model multicatalytic endopeptidase complex receptor repaired response time use

项目摘要

Immunoglobulin (Ig) genes undergo three genetic modifications during B cell development, namely V(D)J recombination, somatic hypermutation, and class switching. For these reactions to occur, RAG and AID enzymes must gain access to recombination and hypermutation sites. Accessibility appears to be provided by the mechanism of gene transcription, presumably by way of chromatin remodeling, which exposes the Ig genes to RAG and AID activity. However, while the accessibility model provides a rationale to specific targeting, it is unclear how DNA lesions downstream of RAG and AID can be processed in the presence of active transcription. Plausibly RNA polymerases could interfere with recombination and hypermutation by transcribing across DNA lesions. We hypothesized therefore that a cellular mechanism must exist that regulates RNA polymerases near DNA DSBs. UV-mediated DNA lesions, for instance, are known to activate the transcription-coupled repair system which mediates ubiquitination and proteasome degradation of stalled RNA polymerases. In similar fashion, DNA polymerases are blocked as a result of DNA damage during the S phase of the cell cycle. In a manuscript published in Nature (June 2007) we have reported a new pathway that regulates RNA synthesis in response to DNA DSBs. In the study we used ribosomal genes as a model because their copy number and nucleolar distribution provide an ideal system to measure the kinetics of gene expression in living cells. In addition, rRNA synthesis is carried out exclusively by RNA polymerase I, whose activity can be easily monitored in real-time by GFP-tagging and photobleaching technology. Using fluorouridine (FUrd) run-on assays in cells exposed to genotoxic stress (γ-irradiation, laser microirradiation, or etoposide treatment) we showed that the presence of DNA breaks elicits a transient block in Pol I rRNA synthesis. This inhibition however did not result from DNA damage per se, but was mediated by the DNA repair proteins ATM, Nbs1, and MDC1. To elucidate the mechanistic details of rDNA transcriptional arrest we labeled several Pol I subunits with GFP and followed their kinetics in the presence or absence of DNA damage. Mathematical modeling of photobleaching data indicated that the ATM pathway interferes with Pol I initiation complex assembly leading to a progressive displacement of elongating holoenzymes from rDNA. The results were confirmed using time-lapse confocal microscopy and biochemical assays. If this same mechanism applies to polymerase II regulation it could explain at least in part why ATM deficient mice and humans consistently develop chromosomal translocations involving antigen receptor genes undergoing recombination. If ATM is not present to shut down transcription at sites of DNA recombination, RNA polymerases might interfere with the proper processing of DNA ends and thus enhancing aberrant repair including translocations. Our next step will be to investigate this same hypothesis using RNA polymerase II reporter systems.

免疫球蛋白（IG）基因在B细胞发育过程中经历了三种遗传修饰，即V（d）J重组，体细胞超突变和类切换。为了使这些反应发生，抹布和辅助酶必须获得重组和超成名位点。可访问性似乎是通过基因转录机理提供的，大概是通过染色质重塑的方式来提供的，这将Ig基因暴露于抹布和辅助活性。但是，虽然可访问性模型为特定靶向提供了理由，但尚不清楚如何在有活动转录的情况下处理抹布和辅助的DNA病变。合理的RNA聚合酶可以通过跨DNA病变转录来干扰重组和超数。我们假设必须存在一种细胞机制，以调节DNA DSB附近的RNA聚合酶。例如，已知紫外线介导的DNA病变会激活转录偶联修复系统，该修复系统介导了失速的RNA聚合酶的泛素体和蛋白酶体降解。以类似的方式，在细胞周期的S相中，DNA损伤阻塞了DNA聚合酶。在自然界发表的手稿中（2007年6月），我们报道了一种新的途径，该途径调节RNA合成以响应DNA DSB。在研究中，我们使用核糖体基因作为模型，因为它们的拷贝数和核仁分布提供了一个理想的系统来测量活细胞中基因表达的动力学。此外，RRNA合成仅由RNA聚合酶I进行，RNA聚合酶I可以通过GFP-Tagging和Photoblaching Technology实时监测其活性。使用暴露于遗传毒性应激的细胞（γ-辐照，激光微辐照或依托泊苷治疗）中的氟环（FURD）跑步测定，我们表明DNA的存在使DNA的存在引起了POL IRRNA合成中的短暂块。然而，这种抑制不是由DNA损伤本身引起的，而是由DNA修复蛋白ATM，NBS1和MDC1介导的。为了阐明RDNA转录停滞的机械细节，我们将几个Pol I亚基标记为GFP，并在存在或没有DNA损伤的情况下跟随其动力学。光漂白数据的数学建模表明，ATM途径会干扰Pol I启动复合物组件，从而导致逐渐延长rDNA的全酶的逐步位移。使用延时共聚焦显微镜和生化测定确认结果。如果这种相同的机制适用于聚合酶II调节，它至少可以解释ATM缺乏小鼠和人类始终如一地发展涉及重组的抗原受体基因的染色体易位。如果不存在ATM以关闭DNA重组部位的转录，则RNA聚合酶可能会干扰DNA末端的正确处理，从而增强包括易位的异常修复。我们的下一步将是使用RNA聚合酶II报告基因系统研究相同的假设。