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. 1982 Sep 25;10(18):5621–5635. doi: 10.1093/nar/10.18.5621

Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity.

H D Riedel, H König, H Stahl, R Knippers
PMCID: PMC320911  PMID: 7145711

Abstract

Unfractionated protein extracts from activated Xenopus laevis eggs contain all functions required for the chain elongation reactions in replicative DNA synthesis (A.Richter, B.Otto and R.Knippers, 1981, Nucl.Ac.Res. 9, 3793-3807). In order to further explore the DNA synthesizing capacity of this in vitro system and to obtain information on the DNA priming activity in these extracts single stranded phage M13-DNA was used as template for in vitro DNA synthesis. The main results of this investigation are: (i) single stranded circular template DNA is converted to a double stranded DNA form in an alpha-amanitin-insensitive reaction which is absolutely dependent on ribonucleoside triphosphates; (ii) the in vitro synthesized complementary strands are DNA fragments of 1000-2000 nucleotides lengths; (iii) the DNA primase activity copurifies through several column steps and sucrose gradient centrifugation with a DNA polymerase alpha. These activities may therefore be closely associated in a quarternary enzyme complex.

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Selected References

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

  1. BALDWIN R. L., SHOOTER E. M. THE ALKALINE TRANSITION OF BU-CONTAINING DNA AND ITS BEARING ON THE REPLICATION OF DNA. J Mol Biol. 1963 Nov;7:511–526. doi: 10.1016/s0022-2836(63)80098-4. [DOI] [PubMed] [Google Scholar]
  2. Benbow R. M., Krauss M. R., Reeder R. H. DNA synthesis in a multi-enzyme system from Xenopus laevis eggs. Cell. 1978 Feb;13(2):307–318. doi: 10.1016/0092-8674(78)90199-x. [DOI] [PubMed] [Google Scholar]
  3. Carrara G., Gattoni S., Mercanti D., Tocchini-Valentini G. P. Purification of a DNA-binding protein from Xenopus laevis unfertilized eggs. Nucleic Acids Res. 1977 Aug;4(8):2855–2870. doi: 10.1093/nar/4.8.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conaway R. C., Lehman I. R. A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2523–2527. doi: 10.1073/pnas.79.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fox A. M., Breaux C. B., Benbow R. M. Intracellular localization of DNA polymerase activities within large oocytes of the frog, Xenopus laevis. Dev Biol. 1980 Nov;80(1):79–95. doi: 10.1016/0012-1606(80)90500-x. [DOI] [PubMed] [Google Scholar]
  6. Gurdon J. B., Birnstiel M. L., Speight V. A. The replication of purified DNA introduced into living egg cytoplasm. Biochim Biophys Acta. 1969 Feb 18;174(2):614–628. doi: 10.1016/0005-2787(69)90291-3. [DOI] [PubMed] [Google Scholar]
  7. Harland R. M., Laskey R. A. Regulated replication of DNA microinjected into eggs of Xenopus laevis. Cell. 1980 Oct;21(3):761–771. doi: 10.1016/0092-8674(80)90439-0. [DOI] [PubMed] [Google Scholar]
  8. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  9. Knippers R., Hoffmann-Berling H. A coat protein from bacteriophage fd. I. Hydrodynamic measurements and biological characterization. J Mol Biol. 1966 Nov 14;21(2):281–292. doi: 10.1016/0022-2836(66)90099-4. [DOI] [PubMed] [Google Scholar]
  10. Knippers R., Razin A., Davis R., Sinsheimer R. L. The process of infection with Bacteriophage phi-X174. XXIX. In vivo studies on the synthesis of the single-stranded DNA of progeny phi-X174 bacteriophage. J Mol Biol. 1969 Oct 28;45(2):237–263. doi: 10.1016/0022-2836(69)90103-x. [DOI] [PubMed] [Google Scholar]
  11. Laskey R. A., Gurdon J. B. Induction of polyoma DNA synthesis by injection into frog-egg cytoplasm. Eur J Biochem. 1973 Sep 3;37(3):467–471. doi: 10.1111/j.1432-1033.1973.tb03007.x. [DOI] [PubMed] [Google Scholar]
  12. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  13. Mattoccia E., Attardi D. G., Tocchini-Valentini G. P. DNA-relaxing activity and endonuclease activity in Xenopus laevis oocytes. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4551–4554. doi: 10.1073/pnas.73.12.4551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Muzyczka N., Poland R. L., Bessman M. J. Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. J Biol Chem. 1972 Nov 25;247(22):7116–7122. [PubMed] [Google Scholar]
  15. Ogawa T., Okazaki T. Discontinuous DNA replication. Annu Rev Biochem. 1980;49:421–457. doi: 10.1146/annurev.bi.49.070180.002225. [DOI] [PubMed] [Google Scholar]
  16. Otto B., Baynes M., Knippers R. A single-strand-specific DNA-binding protein from mouse cells that stimulates DNA polymerase. Eur J Biochem. 1977 Feb 15;73(1):17–24. doi: 10.1111/j.1432-1033.1977.tb11287.x. [DOI] [PubMed] [Google Scholar]
  17. Richter A., Otto B., Knippers R. Replication of SV40 chromatin in extracts from eggs of Xenopus laevis. Nucleic Acids Res. 1981 Aug 11;9(15):3793–3807. doi: 10.1093/nar/9.15.3793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Richter A., Scheu R., Otto B. Replication of simian virus 40 chromatin in vitro depends on the amount of DNA polymerase alpha associated with replicating chromatin. Eur J Biochem. 1980 Aug;109(1):67–73. doi: 10.1111/j.1432-1033.1980.tb04768.x. [DOI] [PubMed] [Google Scholar]
  19. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]

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