Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon, Pyrococcus furiosus.
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 PolTheta, 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.
Fujikane
Ryosuke
R
Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka-shi, Fukuoka 812-8581, Japan.
Komori
Kayoko
K
Shinagawa
Hideo
H
Ishino
Yoshizumi
Y
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology*; D004256:DNA Polymerase I / Q000737:chemistry; D045643:DNA Repair Enzymes; D019641:DNA, Archaeal / Q000737:chemistry*; D045566:DNA, Cruciform / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000737:chemistry; D004331:Drosophila melanogaster; D029968:Escherichia coli Proteins; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D012984:Software; D013696:Temperature; D000097462:DNA Polymerase theta
D000255:Adenosine Triphosphate; D004265:DNA Helicases; D004256:DNA Polymerase I; D045643:DNA Repair Enzymes; D019641:DNA, Archaeal; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C101782:DNApol-theta protein, Drosophila; D000097462:DNA Polymerase theta; C071059:Holliday junction DNA helicase, E coli; C451406:HELQ protein, human