Genetic Analysis Of Xpb Gene In Sulfolobus Islandicus

Nucleotide excision repair (NER) pathway is one of the most important pathways for DNA repair. Comparative genome analyses reveal that while most archaeal organisms have a eukaryotic-like NER pathway, with the XPB, XPD, XPF and XPG proteins, they lack the homologues of many proteins required for DNA repair in Eukarya, especially those involved in regulatory circuits. Therefore, the archaeal NER system is considered to mainly consist of the essential and key components of the eukaryotic system, and thus is regarded as simplified model for studying the more complex eukaryotic NER process. In hyperthermophilic Archaea, gene disruption methods and genetic tools have been developed for the crenarchaeon Sulfolobus islandicus.To investigate the archaeal NER pathway in vivo, we delete the genes encoding the potential NER helicases in the genome of the hyperthermophilic crenarchaeon Sulfolobus islandicus via "maker insertion and unmarkerd target gene deletion" and succeed in isolating deletion mutants of the xpb1 and xpb2 genes, indicating their non-essentiality to S. islandicus survival. Flow cytometry analysis of gene deletion mutants shows that the xpb2 mutant has the same cell cycle with the host strain E233S(ΔpyrEF AlacS), but a number of cells in the population of the xpbl mutant contains more than 2×DNA content, indicating that XPB1 protein not only participates in DNA repair, but also in DNA replication. Phenotypic analyses reveal that the xpb1 mutant is only slightly sensitive to 4-NQO, as compared with the wild-type strain REY15A, while the xpb2 mutant shows no sensitivity to 4-NQO, suggesting the redundancy of NER helicases in S. islandicus. Our work is the first genetic analysis of the crenarchaeon NER pathway.