Repair of localized DNA damage

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，也是一些化疗药物治疗的结果。补骨脂素是一种具有光活性的DNA链间交联剂，已在临床上应用多年。我们合成并证明了抗原连接的补骨脂素的活性。用该化合物孵育的细胞中定义的亚核区域的激光光激活导致局部交联。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跟踪蛋白质进入交联修复位点的招募。在两个循环过程中修复链间交联。在第一个循环中，在交联碱基的两侧切割一条链。在第二个循环中，剩余的内合（仍然交联）碱通过常规的NER去除。除了已经确定的S期修复外，修复是否可以发生在G1期还不确定。我们已经证明，交联在细胞周期的G1期被修复，这一过程依赖于NER功能。XPC蛋白被迅速招募到交联和单加合物的位点。然而，XPE损伤结合复合物被快速招募到单加合物而缓慢招募到交联物。XPE复合物的募集依赖于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，当剩余的单内合碱基被挤出螺旋时，XPE复合物被招募。XPE复合体的募集标志着第一个修复周期的完成和第二个修复周期的开始。

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.