Repair of localized DNA damage

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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.