Characterization of a functional DnaG-type primase in archaea: implications for a dual-primase system.
We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso) and compared in vitro priming kinetics with those of the eukaryotic-type primase (PriSL) also found in Sso. SsoDnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominate primer length was 13 bases, although less abundant products are present. SsoDnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant. Mutation of the conserved glutamate in the active site severely inhibited priming activity, suggesting a functional homology with E. coli DnaG. SsoDnaG was also found to have a greater than fourfold faster rate of DNA priming over that of SsoPriSL under optimal in vitro conditions. The presence of both enzymatically functional primase families in archaea suggests that the DNA priming role may be shared on leading or lagging strands during DNA replication.
Zuo
Zhongfeng
Z
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Rodgers
Cory J
CJ
Mikheikin
Andrey L
AL
Trakselis
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D056890:Eukaryota / Q000201:enzymology; D005454:Fluorescence Polarization; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D018698:Glutamic Acid; D019915:DNA Primase