title: A superfamily 3 DNA helicase encoded by plasmid pSSVi from the hyperthermophilic archaeon Sulfolobus solfataricus unwinds DNA as a higher-order oligomer and interacts with host primase.
abstract: Replication proteins encoded by nonconjugative plasmids from the hyperthermophilic archaea of the order Sulfolobales show great diversity in amino acid sequence. We have biochemically characterized ORF735, a replication protein from pSSVi, an integrative nonconjugative plasmid from Sulfolobus solfataricus P2. We show that ORF735 is a DNA helicase of superfamily 3. It unwound double-stranded DNA (dsDNA) in a 3'-to-5' direction in the presence of ATP over a wide range of temperatures, from 37 degrees C to 75 degrees C, and possessed DNA-stimulated ATPase activity. ORF735 existed in solution as a salt-stable dimer and was capable of assembling into a salt-sensitive oligomer that was significantly larger than a hexamer in the presence of a divalent cation (Mg(2+)) and an adenine nucleotide (ATP, dATP, or ADP) or its analog (ATPgammaS or AMPPNP). Both N-terminal and C-terminal portions of ORF735 (87 and 160 amino acid residues, respectively, in size) were required for protein dimerization but dispensable for the formation of the higher-order oligomer. The protein unwound DNA only as a large oligomer. Yeast two-hybrid and coimmunoprecipitation assays revealed that ORF735 interacted with the noncatalytic subunit of host primase. These findings provide clues to the functional role of ORF735 in pSSVi DNA replication.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Guo
authors.forename: Li
authors.initials: L
authors.lastname: Huang
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism*; D019281:Dimerization; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D016415:Sequence Alignment; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D020798:Two-Hybrid System Techniques
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D000255:Adenosine Triphosphate; D019915:DNA Primase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases