Transcription, Chromatin and DNA repair

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、体细胞超突变和类别转换。要发生这些反应，RAG和AID酶必须能够进入重组和高突变位点。可及性似乎是由基因转录机制提供的，可能是通过染色质重塑的方式，使Ig基因暴露于RAG和AID活性。然而，虽然可及性模型为特定靶向提供了理论基础，但尚不清楚在存在活跃转录的情况下，RAG和AID下游的DNA损伤如何处理。看似合理的是，RNA聚合酶可以通过跨DNA损伤转录来干扰重组和超突变。因此，我们假设一定存在一种细胞机制来调节DNA双链断裂附近的RNA聚合酶。例如，已知紫外线介导的DNA损伤可以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。在细胞周期的S阶段，由于DNA损伤，DNA聚合酶也以类似的方式被阻止。 在《自然》杂志(2007年6月)发表的一篇手稿中，我们报道了一种调节RNA合成的新途径，以响应DNADSB。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为测量活细胞中基因表达的动力学提供了一个理想的系统。此外，rRNA的合成完全由RNA聚合酶I完成，其活性可以通过GFP标记和光漂白技术轻松地实时监测。在暴露于遗传毒性应激(辐射、激光显微照射或依托泊苷治疗)的细胞中，使用氟尿嘧啶(FURD)连续分析，我们发现DNA断裂的存在引起Pol I rRNA合成的一过性阻断。然而，这种抑制并不是由于DNA本身的损伤，而是由DNA修复蛋白ATM、Nbs1和MDC1介导的。为了阐明rDNA转录抑制的机制细节，我们用GFP标记了几个Pol I亚基，并在DNA损伤存在或不存在的情况下跟踪了它们的动力学。光漂白数据的数学模拟表明，ATM途径干扰了Pol I启动复合体的组装，导致延长的全酶从rDNA中逐渐置换。这一结果得到了时间推移共聚焦显微镜和生化分析的证实。 如果同样的机制适用于聚合酶II的调控，它至少可以部分解释为什么ATM缺陷小鼠和人类持续发生涉及抗原受体基因重组的染色体易位。如果不存在ATM来关闭DNA重组位点的转录，RNA聚合酶可能会干扰DNA末端的正确处理，从而增强包括易位在内的异常修复。我们的下一步将是使用RNA聚合酶II报告系统来研究这一假设。

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.