Novel interaction of the bacterial-Like DnaG primase with the MCM helicase in archaea.
DNA priming and unwinding activities are coupled within bacterial primosome complexes to initiate synthesis on the lagging strand during DNA replication. Archaeal organisms contain conserved primase genes homologous to both the bacterial DnaG and archaeo-eukaryotic primase families. The inclusion of multiple DNA primases within a whole domain of organisms complicates the assignment of the metabolic roles of each. In support of a functional bacterial-like DnaG primase participating in archaeal DNA replication, we have detected an interaction of Sulfolobus solfataricus DnaG (SsoDnaG) with the replicative S. solfataricus minichromosome maintenance (SsoMCM) helicase on DNA. The interaction site has been mapped to the N-terminal tier of SsoMCM analogous to bacterial primosome complexes. Mutagenesis within the metal binding site of SsoDnaG verifies a functional homology with bacterial DnaG that perturbs priming activity and DNA binding. The complex of SsoDnaG with SsoMCM stimulates the ATPase activity of SsoMCM but leaves the priming activity of SsoDnaG unchanged. Competition for binding DNA between SsoDnaG and SsoMCM can reduce the unwinding ability. Fluorescent gel shift experiments were used to quantify the binding of the ternary SsoMCM-DNA-SsoDnaG complex. This direct interaction of a bacterial-like primase with a eukaryotic-like helicase suggests that formation of a unique but homologous archaeal primosome complex is possible but may require other components to stimulate activities. Identification of this archaeal primosome complex broadly impacts evolutionary relationships of DNA replication.
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, 801 Chevron, Pittsburgh, PA 15260, USA.
Robert J
RJ
Bauer
Brian W
BW
Graham
Michael A
MA
Trakselis
D004265:DNA Helicases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D019915:DNA Primase / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008954:Models, Biological; D008956:Models, Chemical; D011485:Protein Binding; D025941:Protein Interaction Mapping; D048229:Sulfolobus solfataricus / Q000201:enzymology*
D019641:DNA, Archaeal; D019915:DNA Primase; D004265:DNA Helicases