Abstract:
The human nucleotide excision repair (NER) pathway resolves a range of lesions in DNA including those caused by harmful UV light, environmental mutagens, and agents of chemotherapy. The pathway involves the concerted action of over 30 proteins that recognize the damage, excise it within an oligonucleotide, and fill in the resulting gap, restoring the DNA to its original form. The importance of NER is evident in impaired patients who suffer from a range of diseases with symptoms such as high predisposition to skin cancer, extreme sensitivity to sunlight, and neurological and developmental abnormalities. While the early steps in NER involving the damage recognition and incision steps are well understood, a lot remains to be learned about the later steps including the transition from dual incision to repair synthesis, and finally ligation of the DNA. XPG is the endonuclease that makes the 3' cut on the damaged DNA strand during dual incision. The protein has been suggested to play an important role in regulating the late steps of NER, as it interacts with the replication and repair synthesis factor PCNA. In this thesis the roles of four domains of XPG in the late steps of NER were analyzed. These domains include a previously identified PCNA-interacting domain (PIP-C), a new putative PCNA-interacting domain (PIP-N), a ubiquitin-binding motif (UBM), and the nuclease active site. The nuclease active site mutant (E791A) supported uncoupled incisions as well as partial repair synthesis, suggesting that the incisions could be ordered with the 5' incision occurring before the 3' incision, and that XPG itself is involved in regulating the transition from dual incision to repair synthesis. Additionally, the PIP-N and UBM domains were found to be important for damage removal, as well as assembly and disassembly of late factors of NER. The UBM mutant displayed a phenotype similar to the E791A mutant, suggesting that an interaction of XPG with ubiquitin is important to trigger its nuclease activity. This work reveals that the timing of the XPG incision plays an important role in regulating the late steps of NER, and that interactions with PCNA and ubiquitin are important elements of this regulation.
