Repair of localized DNA damage

未能有效应对复制压力被认为是发育缺陷的关键因素、过早衰老综合征的基础以及肿瘤发展的刺激因素。链间交联是特别危险的 DNA 损伤，因为它们绝对阻碍复制，因此是复制应激反应的主要挑战。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。补骨脂素是光活性 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补骨脂素，并证明了其活性。 与化合物一起孵育的细胞中确定的亚核区域的激光光活化导致局部交联。 在修复良好和缺陷的细胞中监测这些加合物的修复。我们正在使用这种方法来跟踪蛋白质招募到交联修复位点的情况。链间交联在两个循环过程中修复。在第一个循环中，在交联碱基的两侧切割链上。在第二个循环中，通过传统的 NER 去除剩余的加合（且仍交联）碱基。除了成熟的S期修复之外，G1期是否可以发生修复还存在不确定性。我们已经证明交联在细胞周期的 G1 期修复，这一过程依赖于 NER 功能。 XPC 蛋白被快速招募到交联和单加合物位点。 然而，XPE 损伤结合复合物被快速募集成单加合物，并缓慢地交联。 XPE 复合物的募集取决于 XPC 活性和修复合成。我们的结果支持这样一种情况，其中 XPE 复合物不识别交联，但在第一个修复周期完成后，当剩余的单加合碱基被迫离开螺旋时，XPE 复合物会被招募。 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。 我们已将该技术应用于检查 FancD2 在 ICL 修复中的功能。 FancD2 蛋白是范可尼贫血途径的中心节点。该途径存在缺陷的个体会遭受严重的发育缺陷，并在产后生活中表现出过早衰老的迹象。该途径在复制应激的反应中发挥着关键作用。 我们发现 FancD2 以独立于细胞周期的方式被招募到多个刺激双链断裂中；激光局部补骨脂素交联与细胞周期无关；并仅在 S 相中进行激光局部交联。了解范可尼途径缺陷的性质将为开发这种疾病的有效疗法奠定基础。 我们还描述了 Fan1 的募集和对 ICL 修复的贡献，Fan1 是最近发现的与 FancD2 相关的核酸酶。 目前认为 Fan1 向 ICL 的招募依赖于 FancD2。然而，我们发现该蛋白以不依赖 FancD2 的方式快速招募至 ICL。第二波积累取决于与 FancD2 的关联。我们现在正在对该蛋白质的不同结构域在 ICL 响应中的作用进行结构功能分析。

Failure to respond effectively to replication stress is recognized as a key contributor to developmental defects, the basis of premature aging syndromes, and a stimulus for the development of neoplasia. Interstrand crosslinks are particularly dangerous DNA lesions as they are absolute blocks to replication, and thus a major challenge to the replication stress response. 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 have 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 monitored 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. We have applied this technology to an examination of the function of FancD2 in ICL repair. The FancD2 protein is the central node in the Fanconi Anemia pathway. Individuals with deficiencies in this pathway suffer severe developmental defects, and show signs of premature aging during postpartum life. The pathway plays a key role in the response to replication stress. We find that FancD2 is recruited to multiple stimuli-double strand breaks in a cell cycle independent manner; laser localized psoralen crosslinks independent of cell cycle; and to laser localized crosslinks only in S phase. Understanding the nature of defects in the Fanconi pathway will provide the basis for developing effective therapies for this disorder. We have also characterized the recruitment and contribution to ICL repair of Fan1, a recently discovered nuclease that associates with FancD2. It is currently believed that Fan1 recruitment to ICLs is dependent on FancD2. However we have found that this protein is rapidly recruited to ICLs, in a FancD2 independent manner. There is a second wave of accumulation that is dependent on the association with FancD2. We are now engaged in a structure function analysis of the role of different domains of this protein in the response to ICLs.