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.
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.
Guo
Xin
X
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
Huang
Li
L
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
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D000255:Adenosine Triphosphate; D019915:DNA Primase; D000251:Adenosine Triphosphatases; D004265:DNA Helicases