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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三种遗传修饰，即V（D）J重组、体细胞超突变和类转换。为了使这些反应发生，RAG和AID酶必须能够进入重组和超突变位点。可接近性似乎是由基因转录机制提供的，推测是通过染色质重塑的方式，其使IG基因暴露于RAG和AID活性。然而，虽然可及性模型为特异性靶向提供了理论基础，但尚不清楚在存在活性转录的情况下如何处理RAG和AID下游的DNA损伤。RNA聚合酶可能通过跨DNA损伤转录来干扰重组和超突变。因此，我们假设一定存在调节DNA双链断裂附近RNA聚合酶的细胞机制。例如，已知UV介导的DNA损伤激活转录偶联修复系统，该系统介导停滞的RNA聚合酶的泛素化和蛋白酶体降解。以类似的方式，DNA聚合酶在细胞周期的S期由于DNA损伤而被阻断。在《自然》杂志（2007年6月）上发表的一篇论文中，我们报道了一种新的调节RNA合成的途径。在这项研究中，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，其活性可以通过GFP标记和光漂白技术容易地实时监测。使用氟尿苷（FURD）运行在细胞暴露于遗传毒性应激（辐射，激光照射，或依托泊苷治疗）的测定，我们表明，DNA断裂的存在eldamn一个短暂的块在Pol I rRNA合成。γ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MDC 1介导的。为了阐明rDNA转录停滞的机制细节，我们用GFP标记了几个Pol I亚基，并在存在或不存在DNA损伤的情况下跟踪它们的动力学。光漂白数据的数学建模表明，ATM途径干扰Pol I起始复合物的组装，导致从rDNA延伸全酶的逐步位移。使用延时共聚焦显微镜和生物化学测定证实了结果。如果这种相同的机制适用于聚合酶II的调节，它可以至少部分解释为什么ATM缺陷小鼠和人类始终发展染色体易位涉及抗原受体基因进行重组。如果ATM不存在以关闭DNA重组位点处的转录，则RNA聚合酶可能干扰DNA末端的正确加工，从而增强异常修复，包括易位。我们的下一步将是使用RNA聚合酶II报告系统来研究这一相同的假设。