Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end.
Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate. These activities depend on MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).
Higashibata
Hiroki
H
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan.
Kikuchi
Hisasi
H
Kawarabayasi
Yutaka
Y
Matsui
Ikuo
I
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D019843:Archaeal Proteins; D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D003851:Deoxyribonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae