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、体细胞超突变和类转换。为了使这些反应发生，RAG和AID酶必须进入重组和高突变位点。可接近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异性靶向提供了理论依据，但尚不清楚在活性转录存在的情况下，RAG和AID下游的DNA损伤如何被处理。似乎RNA聚合酶可以通过转录DNA病变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 dsb附近的RNA聚合酶。例如，已知紫外线介导的DNA损伤会激活转录偶联修复系统，该系统介导泛素化和蛋白酶体降解停滞的RNA聚合酶。类似地，DNA聚合酶在细胞周期的S期由于DNA损伤而被阻断。

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.