In most organisms, DNA replication is initiated by DNA primases, which 
synthesize primers that are elongated by DNA polymerases. In this study, we 
describe the isolation and biochemical characterization of the DNA primase 
complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. 
kodakaraensis DNA primase complex is a heterodimer containing stoichiometric 
levels of the p41 and p46 subunits. The catalytic activity of the complex 
resides within the p41 subunit. We show that the complex supports both DNA and 
RNA synthesis, whereas the p41 subunit alone marginally produces RNA and 
synthesizes DNA chains that are longer than those formed by the complex. We 
report that the T. kodakaraensis primase complex preferentially interacts with 
dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA 
chains de novo. The latter findings indicate that the archaeal primase complex, 
in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the 
absence of ribonucleoside triphosphates. DNA primers formed by the archaeal 
complex can be elongated extensively by the T. kodakaraensis DNA polymerase 
(Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. 
Supplementation of reactions containing the p41 subunit with the p46 subunit 
leads to PolB-catalyzed DNA synthesis. We also established a rolling circle 
reaction using a primed 200-nucleotide circle as the substrate. In the presence 
of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, 
PolB, replication factor C, and proliferating cell nuclear antigen, long leading 
strands (>10 kb) are produced. Supplementation of such reactions with the DNA 
primase complex supported lagging strand formation as well.