Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Sep 24;11(18):6369–6380. doi: 10.1093/nar/11.18.6369

Mechanism of stimulation by a specific protein factor of de novo DNA synthesis by mouse DNA replicase with fd phage single stranded circular DNA.

T Yagura, T Kozu, T Seno
PMCID: PMC326379  PMID: 6622256

Abstract

Mouse DNA replicase is a functional multienzyme complex consisting of DNA polymerase and DNA primase. The DNA and initiator RNA syntheses by DNA replicase with single stranded DNA as template are stimulated by a stimulating factor (T. Yagura, T. Kozu and T. Seno, 1982, J. Biochem. (Tokyo).91, 607-618). The action mechanism of the stimulating factor on this novel DNA synthesis with fd phage single stranded circular DNA as template was studied. The stimulating factor directly stimulated initiator RNA synthesis but did not change the length of either initiator RNA (8 to 10 nucleotides long) or the product DNA (300 to 1,000 nucleotides long). Kinetic studies and analysis of the products by neutral agarose gel electrophoresis show that the stimulating factor increased the affinity of DNA replicase for template DNA without changing the apparent Km values for deoxy- and ribonucleotide substrates. Thus, in combination with a sufficient amount of the stimulating factor, DNA replicase quantitatively converted the template DNA to the position of double-stranded circular replicative form II DNA, as shown by agarose gel electrophoresis.

Full text

PDF
6369

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beck E., Sommer R., Auerswald E. A., Kurz C., Zink B., Osterburg G., Schaller H., Sugimoto K., Sugisaki H., Okamoto T. Nucleotide sequence of bacteriophage fd DNA. Nucleic Acids Res. 1978 Dec;5(12):4495–4503. doi: 10.1093/nar/5.12.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  3. Kozu T., Aida M., Ikawa Y., Seno T. Analysis of synthetic RNA-directed DNA polymerase activities in the spleen of a rat with myelogenous leukemia induced by N-nitroso-N-butylurea. Eur J Cancer. 1978 Oct;14(10):1065–1076. doi: 10.1016/0014-2964(78)90062-2. [DOI] [PubMed] [Google Scholar]
  4. Kozu T., Yagura T., Seno T. De novo DNA synthesis by a novel mouse DNA polymerase associated with primase activity. Nature. 1982 Jul 8;298(5870):180–182. doi: 10.1038/298180a0. [DOI] [PubMed] [Google Scholar]
  5. Nishizawa M., Tanabe K., Matsukage A., Nakamura H., Yagura T. Detection of DNA replicase activity in rat thymoma cells. J Biochem. 1983 Jan;93(1):287–290. doi: 10.1093/oxfordjournals.jbchem.a134165. [DOI] [PubMed] [Google Scholar]
  6. Sugiura M., Okamoto T., Takanami M. Starting nucleotide sequences of RNA synthesized on the replicative form DNA of coliphage fd. J Mol Biol. 1969 Jul 28;43(2):299–315. doi: 10.1016/0022-2836(69)90269-1. [DOI] [PubMed] [Google Scholar]
  7. Villani G., Fay P. J., Bambara R. A., Lehman I. R. Elongation of RNA-primed DNA templates by DNA polymerase alpha from Drosophila melanogaster embryos. J Biol Chem. 1981 Aug 10;256(15):8202–8207. [PubMed] [Google Scholar]
  8. Yagura T., Kozu T., Seno I., Nishijima T., Ohtsuka E., Ikehara M. Inhibition of a novel subspecies of DNA polymerase alpha by 2'-deoxy-2'-azidoadenosine 5'-triphosphate. Biochem Biophys Res Commun. 1982 Feb 26;104(4):1189–1195. doi: 10.1016/0006-291x(82)91376-6. [DOI] [PubMed] [Google Scholar]
  9. Yagura T., Kozu T., Seno T. Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization. J Biochem. 1982 Feb;91(2):607–618. doi: 10.1093/oxfordjournals.jbchem.a133732. [DOI] [PubMed] [Google Scholar]
  10. Yagura T., Kozu T., Seno T. Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size. J Biol Chem. 1982 Sep 25;257(18):11121–11127. [PubMed] [Google Scholar]
  11. Yagura T., Tanaka S., Kozu T., Seno T., Korn D. Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha. J Biol Chem. 1983 Jun 10;258(11):6698–6700. [PubMed] [Google Scholar]
  12. Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES