Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Abstract

Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand crosslinks. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

“…The helicase and nuclease activities of the purified T. kodakarensis Hef protein (TkoHef) for forkstructured DNA was also confirmed in vitro (5). These genetic and biochemical properties of TkoHef suggested that this protein actually works at stalled replication forks in T. kodakarensis by the coordination of its helicase and endonuclease activities, as predicted.…”

“…Production and Purification of TkoHef-Cloning of the hef gene and preparation of the TkoHef protein were described in detail in our previous report (5). For the preparation of the His-tagged IDR (His-ID), the gene fragment encoding Met 490 -Lys 593 of TkoHef (Hef-ID) was amplified by PCR from T. kodakarensis DNA, using the primer set ID-F/ID-R including NdeI and NotI recognition sequences at their 5Ј-regions (shown in Table 1) for insertion into the NdeI-NotI sites of a modified pET-28a(ϩ) vector (Novagen).…”

“…A genetic manipulation system was developed for Thermococcus kodakarensis (4), and phenotype analyses of the hef mutant strain confirmed that Hef is involved in multiple repair processes, and its especially high sensitivity to mitomycin C suggested that Hef performs a critical function in DNA interstrand cross-link repair (5). The helicase and nuclease activities of the purified T. kodakarensis Hef protein (TkoHef) for forkstructured DNA was also confirmed in vitro (5).…”

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

“…The ssDNAbinding proteins of euryarchaeotes resemble eukaryotic replication proteins A (RPA)s whereas those of crenarchaeotes resemble bacterial single-stranded binding proteins (SSBs) (Kerr et al, 2003;Rolfsmeier & Haseltine, 2010). Furthermore, genes for several HR proteins which can be deleted from bacteria and eukaryotes are essential in hyperthermophilic archaea (Fujikane et al, 2010;Zhang et al, 2013), suggesting that, in archaea, these proteins play unusually important roles.…”

Archaeal DNA Repair Nucleases

“…In support to this, a recent genetic analysis of hef mutants in the hyperthermophilic archaeon, Thermococcus Kodakaraensis, has demonstrated that Hef is involved in the repair of a wide variety of DNA damages. In addition, the higher sensitivity of hef mutants to methyl sulfonate and mitomycin C, suggests a central role for Hef protein in the archaeal NER and/or ICL repair pathways (Fujikane et al, 2010).…”