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。与化合物孵育的细胞中特定的亚核区域的激光光激活导致了局部的交联键。这些加合物的修复是在修复熟练和缺陷的细胞中进行的。我们正在使用这种方法来跟踪蛋白质在交联链修复部位的招募情况。链间交联链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On链。在第二个循环中，通过传统的NER除去剩余的加成碱(并且仍然是交联碱)。除了公认的S期修复外，是否能在G1期修复还存在不确定性。我们已经证明，在细胞周期的G1期，交联链被修复，这一过程依赖于NER功能。XPC蛋白被迅速募集到交联点和单加合物位点。然而，XPE损伤结合络合物被迅速地招募到单加合物中，并缓慢地被招募到交联物中。XPE复合体的募集取决于XPC的活性和修复合成。我们的结果支持这样一种情况，即XPE复合体不识别交联键，但在第一个修复周期完成后，当剩余的单加成碱基被迫离开螺旋时，XPE复合体被招募。XPE建筑群的启用标志着第一个修复周期的完成和第二个修复周期的开始。 我们还检测了FANCD2蛋白的募集动态，FANCD2蛋白是Fanconi贫血途径的中心节点。我们发现，FANCD2以不依赖于细胞周期的方式被三种刺激-双链断裂招募；激光定位的补骨脂素交联物，依赖于XPC，但不依赖于细胞周期；激光定位的交联物以XPC不依赖的方式，仅在S时相。了解Fanconi通路缺陷的本质将为开发有效的治疗该疾病的方法提供基础。

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. We have also examinedd the recruitment dynamics of the FancD2 protein, the central node in the Fanconi Anemia pathway. We find that FancD2 is recruited to three kinds of stimuli-double strand breaks in a cell cycle independent manner; laser localized psoralen crosslinks, XPC dependent, but independent of cell cycle; and to laser localized crosslinks in an XPC independent fashion, only in S phase. Understanding the nature of defects in teh Fanconi pathway will provide the basis for developing effective therapies for this disorder.