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Characterization of 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 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.

SUBMITTER: Chemnitz Galal W

PROVIDER: S-EPMC3351342 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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

Chemnitz Galal Wiebke W Pan Miao M Kelman Zvi Z Hurwitz Jerard J

The Journal of biological chemistry 20120217 20

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 ...[more]

PMID: 22351771

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Project description:Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-alpha-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-alpha-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an alpha-glucosidase, a p-nitrophenyl-alpha-D-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98 degrees C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60 degrees C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5'-phosphate is conserved in the T. litoralis enzyme.

Dataset Information

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

Publications

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

Similar Datasets

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