Identification of a Novel Helicase Activity Unwinding Branched DNAs from the Hyperthermophilic Archaeon, Pyrococcus furiosus

Abstract

To identify the branch migration activity in archaea, we fractionated Pyrococcus furiosus cell extracts by several chromatography and assayed for ATP-dependent resolution of synthetic Holliday junctions. The target activity was identified in the column fractions, and the optimal reaction conditions for the branch migration activity were determined using the partially purified fraction. We successfully cloned the corresponding gene by screening a heat-stable protein library made by P. furiosus genomic DNA. The gene, hjm (Holliday junction migration), encodes a protein composed of 720 amino acids. The Hjm protein is conserved in Archaea and belongs to the helicase superfamily 2. A homology search revealed that Hjm shares sequence similarity with the human Pol⌰, HEL308, and Drosophila Mus308 proteins, which are involved in a DNA repair, whereas no similar sequences were found in bacteria and yeast. The Hjm helicase may play a central role in the repair systems of organisms living in extreme environments.Homologous recombination plays important roles in DNA metabolisms, including several DNA repair processes and the generation of genetic diversity in living cells. The Holliday junction (HJ) 1 is an important intermediate during the homologous recombination process (1). The molecular mechanism of the early stage of homologous recombination has been extensively investigated, and the proteins involved in this process are conserved in the three biological domains: Bacteria, Eukarya, and Archaea (2, 3). On the other hand, the protein factors involved in the late stage of the process have been identified only in Bacteria, in which the RuvABC-Holliday junction complex processes the recombinational intermediates (4, 5). In Eukarya, the activities for branch migration and resolution of the HJ have been reported (6 -9); however, the corresponding proteins have yet to be identified. A recent report showed that RAD51C, a RAD51 paralog in human cells, is involved in HJ processing (10).Archaea, the third domain of life (11), is distinct from both Bacteria and Eukarya. Archaea share many similarities with Eukarya in their genetic information processing pathways, including DNA replication, transcription, and translation (12), although cellular structure is prokaryotic. Therefore, the archaeal processes provide a useful model systems to understand the much more complex mechanisms of their eukaryotic equivalents. Proteins involved in homologous recombination are also conserved between Archaea and Eukarya. We characterized the Pyrococcus furiosus RadA and RadB proteins (13-15), which play a central role in the initiation of homologous recombination. These archaeal recombinases have sequences more similar to that of eukaryotic Rad51 than bacterial RecA (16,17). In addition, we showed that the P. furiosus RPA, which is composed of three subunits, RPA41, RPA14, and RPA32, like the eukaryotic RPA (p70-p14-p32), but different from bacterial SSB (homotetramer), clearly stimulated a RadA-mediated strand exchange reaction (18). The homologs...

Hjm/Hel308A DNA Helicase fromSulfolobus tokodaiiPromotes Replication Fork Regression and Interacts with Hjc Endonuclease In Vitro

“…Hjm was originally identified by screening cell extracts and the genomic library of the euryarchaeon P. furiosus (8). It has been shown to dissolve four-way junctions in an ATPdependent manner (8).…”

“…There have been a few reports about DNA helicases that are involved in recombination repair in archaea (8,9,11,13) but none regarding helicases that promote fork regression. Two helicases, Holliday junction migration DNA helicase (Hjm) from Pyrococcus furiosus and Hel308a from Methano-thermobacter thermautotrophicus, have been identified recently (8,9,11). However, the real functions of Hjm/Hel308a helicases are unknown.…”

DNA Replication in the Archaea

“…Strikingly similar results were observed with the Pyrococcus homolog, termed Hjm. When expressed in an E. coli strain lacking RecQ (a DNA helicase associated with recovery of stalled replication forks), Hel308a/Hjm complemented the recQ phenotype, strongly suggesting that Hel308a/Hjm may play a similar role in archaea (6,35,42). Interestingly, in some archaea the Hel308a homologue is encoded within an operon-like structure along with MCM and GINS, suggestive of a linked function of these proteins.…”

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

“…The Xpb, Xpd, Xpg, and Hef/Xpf proteins, which are the homologues of the eukaryotic proteins encoded by genes whose mutations result in the human Xeroderma pigmentosum syndrome (XP), may function in the NER pathway as helicases or nucleases in Archaea (Komori et al, 2002(Komori et al, , 2004Roberts et al, 2003;Roberts and White, 2005;Rudolf et al, 2006;Richards et al, 2008;Roth et al, 2009;Rouillon and White, 2010). A biochemical screen to detect an activity that can promote the migration of synthetic Holliday junctions identified the Hjm/Hel308a helicase (Fujikane et al, 2005). This archaeal protein partially complemented the defect of RecQ functions in a recQ mutant Escherichia coli cells, and may be involved in stalled replication fork stabilization like eukaryotic RecQ family proteins including WRN, BLM, and Sgs1 (Fujikane et al, 2006;Guy and Bolt, 2005).…”