Transcription, Chromatin and DNA Repair

转录、染色质和 DNA 修复

• 批准号: 7138040
• 负责人: rafael c casellas
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 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/7138040
• 关键词: DNA binding protein; DNA damage; DNA directed DNA polymerase; DNA repair; chromatin; confocal scanning microscopy; genetic transcription; molecular genetics

The molecular pathways leading to DNA repair upon damage are not well understood. In the immune system, B and T cells undergo a variety of DNA modifying events, such as recombination and hypermutation, which require the activity of a variety of DNA repair enzymes. Both recombination and hypermutation are intermediate mechanisms in the processing of cell surface receptors in B and T cells. These receptors are absolutely required to the proper functioning of lymphocytes. Thus, animals or humans deficient in B and T cell receptors are completely immunodeficienty and succumb to infections quite readily. Unfortunately, DNA damage can also lead to translocations and malignancy in lymphocytes when improperly repaired. For instance, mammals lacking ATM, a key enzyme in the repair of DNA damage in lymphocytes, develop T cell lymphomas early in life. To investigate the molecular pathways involved in DNA repair we are making use of confocal microscopy. By labeling DNA repair enzymes, histones, and DNA polymerases with yellow and cyan fluorescent proteins (YFP and CFP) we are able to visualize the kinetics of DNA repair in living cells. With this new approach, we have identified a new role of ATM in the processing of DNA double-stranded breaks. We found that upon DNA induced damage, ATM quickly downregulates transcription at sites near the lesion. Both in cell lines and primary cells we found that gene transcription continues in the presence of broken DNA. Providing that the transcription machinery interferes with the processing of DNA ends, our results provide a rationale for the impaired recombination and the frequent translocations observed in ATM-/- lymphocytes.

导致DNA损伤后修复的分子途径还不清楚。在免疫系统中，B和T细胞经历多种DNA修饰事件，例如重组和超突变，这需要多种DNA修复酶的活性。重组和超突变都是B和T细胞中细胞表面受体加工的中间机制。这些受体是淋巴细胞正常运作所必需的。因此，缺乏B和T细胞受体的动物或人是完全免疫缺陷的，并且非常容易感染B。不幸的是，DNA损伤也可能导致易位和恶性淋巴细胞修复不当时。例如，缺乏ATM的哺乳动物，在淋巴细胞中修复DNA损伤的关键酶，在生命早期发展T细胞淋巴瘤。 为了研究参与DNA修复的分子途径，我们正在使用共聚焦显微镜。通过用黄色和青色荧光蛋白（YFP和CFP）标记DNA修复酶，组蛋白和DNA聚合酶，我们能够可视化活细胞中DNA修复的动力学。通过这种新方法，我们已经确定了ATM在DNA双链断裂过程中的新作用。我们发现，在DNA诱导的损伤，ATM迅速下调转录在病变附近的网站。在细胞系和原代细胞中，我们发现基因转录在DNA断裂的情况下继续进行。如果转录机制干扰DNA末端的加工，我们的结果为在ATM-/-淋巴细胞中观察到的重组受损和频繁易位提供了依据。