Duan, Zhenhong
(2009)
Genetic analysis of two structure-specific endonucleases Hef and Fen1 in archaeon Haloferax volcanii.
PhD thesis, University of Nottingham.
Abstract
Nucleotide excision repair (NER) is a versatile pathway of DNA repair that deals with a variety of DNA lesions, such as UV-induced DNA damage and interstrand crosslinks. In bacteria, the UvrABC system carries out NER. In human cells, XPF and XPG are two structure-specific endonucleases that act in NER. XPF is responsible for a 5' incision at the DNA lesion and XPG carries out the 3' incision. In Archaea, the third domain of life, most species have homologues of some eukaryal NER proteins. Interestingly, Haloferax volcanii encodes homologues of both the eukaryotic NER genes (XPF, XPG, XPB and XPD) and bacterial NER genes (uvrA, uvrB, uvrC and uvrD). In this study, the function of XPG, XPF and UvrA in H. volcanii is investigated.
XPG is related to FEN1, a structure-specific 5' flap endonuclease that acts in Okazaki fragment maturation. H. volcanii has a single gene homologous to both XPG and FEN1. The helicase/nuclease hef gene in H. volcanii is the archaeal homologue of human XPF, but also shows homology to Mus81 and FANCM. Mus81 has been found to resolve joint molecules in yeast, while FANCM is required for the repair of interstrand crosslinks in vertebrates. The uvrA gene in H. volcanii is the archaeal homologue of bacterial uvrA, which encodes a protein that plays a vital role in NER at the DNA damage recognition step.
This study demonstrates that in H. volcanii, UvrA is involved in the major pathway for repair of UV induced DNA damage. By contrast, Hef and UvrA are involved in two different pathways for the repair of mitomycin C induced DNA crosslinks. Fen1 and Hef have overlapping functions for the repair of DNA cross-links, but not oxidative damage. We also obtain a spontaneous suppressor sfnA, which can suppress the slow growth and MMC sensitivity, but not the UV sensitivity of fen1 deletion mutants.
Using plasmid assays, it has been shown that the hef deletion mutant is deficient in accurate end-joining and homologous recombination, including both crossover and non-crossover recombination. In contrast, Fen1 has no significant role in accurate end-joining, but Fen1 may regulate the ratio of non-crossover recombination to crossover recombination.
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