Characterization of the DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis.

Abstract:

In most organisms, DNA replication is initiated by DNA primases which synthesize primers that are elongated by DNA polymerases. In this report we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis (Tk). The Tk 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 while the p41 subunit alone, marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the Tk primase complex preferentially interacts with dNTP rather than rNTPs 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 rNTPs. DNA primers formed by the archaeal complex can be elongated extensively by the Tk 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 Tk MCM 3'→5' DNA helicase, PolB, RFC and PCNA, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.

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