Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis Journal Article
| Authors: | Galal, W. C.; Pan, M.; Kelman, Z.; Hurwitz, J. |
| Article Title: | 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. |
| Keywords: | controlled study; unclassified drug; nonhuman; dna polymerase; dna replication; dna synthesis; cell proliferation; complex formation; protein protein interaction; rna; dna; rna synthesis; eukaryota; multiprotein complexes; helicase; catalysis; dna replications; proliferating cell nuclear antigens; replication factor c; adenosine triphosphate; cycline; protein subunits; heterodimer; archaea; synthesis (chemical); dna helicase; archaeal proteins; stoichiometry; ribonucleotide; rna, archaeal; dna directed dna polymerase beta; minichromosome maintenance protein 3; biochemical characterization; heterodimers; deoxyribonucleotide; thermococcus kodakarensis; thermococcus; catalytic subunits; microorganisms; dna chains; dna synthesiss; lagging strand; deoxyadenosine triphosphate; dna primase; ribonucleosides; minichromosome; protein p46; dna, archaeal; thermococcus kodakaraensis; protein p41; archaeal; archaeon; archaeon thermococcus kodakaraensis; complex support; dna primase complex |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 287 |
| Issue: | 20 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2012-05-11 |
| Start Page: | 16209 |
| End Page: | 16219 |
| Language: | English |
| DOI: | 10.1074/jbc.M111.338145 |
| PROVIDER: | scopus |
| PMCID: | PMC3351342 |
| PUBMED: | 22351771 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 4 June 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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