Repair of localized DNA damage

修复局部DNA损伤

• 批准号: 7964038
• 负责人: Michael Seidman
• 金额: $ 36.31万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 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/7964038
• 关键词: Antigens; Binding; Cell Cycle; Cell Respiration; Cells; Complex; Crosslinker; DNA; DNA Damage; DNA Interstrand Crosslinking; DNA lesion; Ficusin; G1 Phase; Genetic Transcription; Incubated; Individual; Laser Microscopy; Lasers; Life; Link; Monitor; Pharmaceutical Preparations; Phase; Process; Proteins; Psoralens; Recruitment Activity; Side; Site; Technology; Uncertainty; adduct; base; chemotherapy; crosslink; interest; photoactivation; repaired; response

Interstrand crosslinks are particularly dangerous DNA lesions as they are absolute blocks to replication and transcription. They are believed to occur as a product of oxidative metabolism, and are also a consequence of treatment with some chemotherapy drugs. Psoralens are photoactive DNA interstrand crosslinkers that have been used clinically for many years. We synthesized, and demonstrated the activity of, antigen linked psoralens. Laser photoactivation of defined subnuclear regions in cells incubated with the compounds resulted in localized crosslinks. Repair of these adducts was followed in repair proficient and deficient cells. We are using this approach to follow the recruitment of proteins into sites of crosslink repair. Interstrand crosslinks are repaired in a two cycle process. In the first cycle on strand is incised on either side of the crosslinked base. In the second cycle the remaining adducted (and still crosslinked) base is removed via conventional NER. There is uncertainty as to whether repair can occur in G1 phase, in addition to the well established S phase repair. We have shown that crosslinks are repaired in the G1 phase of the cell cycle, in a process that is dependent on NER functions. XPC protein was rapidly recruited to sites of crosslinks and monoadduct. However, the XPE damage binding complex was recruited rapidly to monoadducts and slowly to crosslinks. Recruitment of the XPE complex was dependent on XPC activity, and repair synthesis. Our results support a scenario in which the XPE complex does not recognize the crosslink, but is recruited when the remaining monoadducted base is forced out of the helix after the completion of the first repair cycle. The recruitment of the XPE complex is a marker of completion of the first repair cycle and the start of the second.

链间交联是特别危险的DNA损伤，因为它们是复制和转录的绝对障碍。它们被认为是氧化代谢的产物，也是某些化疗药物治疗的结果。光活性DNA链间交联剂已在临床上使用多年。我们合成了抗原连接的peptiens，并证明了其活性。 在与化合物孵育的细胞中，激光光活化确定的亚核区域导致局部交联。 这些加合物的修复在修复熟练和缺陷细胞中进行。我们正在使用这种方法来跟踪蛋白质进入交联修复位点的募集。在两个循环过程中修复链间交联。在第一个循环中，在交联基底的任一侧切割股线。在第二个循环中，通过常规NER除去剩余的加合（并且仍然交联）碱。除了已确立的S期修复外，G1期是否会发生修复还存在不确定性。我们已经表明，在依赖于NER功能的过程中，交联在细胞周期的G1期被修复。XPC蛋白被迅速募集到交联和单加合物的位点。 然而，XPE损伤结合复合物被迅速募集为单加合物，并缓慢地募集为交联物。募集的XPE复合物依赖于XPC活性，和修复合成。我们的研究结果支持这样一种情况，即XPE复合物不识别交联，但在完成第一个修复周期后，当剩余的单加合碱基被迫离开螺旋时，XPE复合物被招募。XPE复合物的募集是第一个修复周期完成和第二个修复周期开始的标志。