Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1978 Feb;75(2):605–609. doi: 10.1073/pnas.75.2.605

Analysis of the phiX DNA replication cycle by electron microscopy.

K Koths, D Dressler
PMCID: PMC411304  PMID: 273222

Abstract

We have monitored the development of intracellular phiX DNA forms during the course of a virus life cycle that duplicates as closely as possible the normal infection of individual cells by single virions. The viral DNA was isolated in a one-step purification procedure, and quantitative electron microscopy was performed on the samples, resulting in the following conclusions: (i) Early in the life cycle, when the cells accumulate duplex rings, two types of DNA replication intermediates are observed: a rolling circle with a single-stranded tail; and a novel form, a single-stranded circle that is partially duplex. Thus, duplex ring synthesis appears to occur in two asymmetric steps, with positive strand DNA first being processed from the tail of the rolling circle and circularized, before it acts as a template for negative strand synthesis. (ii) Late in the life cycle, as single-stranded circles are synthesized and virus particles are assembled, only one replicating intermediate is observed--the rolling circle with a single-stranded tail. At this stage, the number of rolling circles reaches a level of about 35 per cell. (iii) The net rate of polymerization in the rolling circle intermediates is about 200 nucleotides per sec.

Full text

PDF
605

Images in this article

607Image
on p.607
608Image
on p.608
608Image
on p.608

Selected References

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

  1. Benbow R. M., Zuccarelli A. J., Sinsheimer R. L. Recombinant DNA molecules of bacteriophage phi chi174. Proc Natl Acad Sci U S A. 1975 Jan;72(1):235–239. doi: 10.1073/pnas.72.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Doniger J., Warner R. C., Tessma I. Role of circular dimer DNA in the primary recombination mechanism of bacteriophage S13. Nat New Biol. 1973 Mar 7;242(114):9–12. doi: 10.1038/newbio242009a0. [DOI] [PubMed] [Google Scholar]
  3. Dressler D. The rolling circle for phiX DNA replication. II. Synthesis of single-stranded circles. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1934–1942. doi: 10.1073/pnas.67.4.1934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dressler D., Wolfson J. The rolling circle for phi X DNA replication. 3. Synthesis of supercoiled duplex rings. Proc Natl Acad Sci U S A. 1970 Sep;67(1):456–463. doi: 10.1073/pnas.67.1.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eisenberg S., Griffith J., Kornberg A. phiX174 cistron A protein is a multifunctional enzyme in DNA replication. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3198–3202. doi: 10.1073/pnas.74.8.3198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenberg S., Scott J. F., Kornberg A. An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174. Proc Natl Acad Sci U S A. 1976 May;73(5):1594–1597. doi: 10.1073/pnas.73.5.1594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fujisawa H., Hayashi M. Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage phi X174. J Virol. 1976 Aug;19(2):409–415. doi: 10.1128/jvi.19.2.409-415.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gellert M., Mizuuchi K., O'Dea M. H., Nash H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3872–3876. doi: 10.1073/pnas.73.11.3872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilbert W., Dressler D. DNA replication: the rolling circle model. Cold Spring Harb Symp Quant Biol. 1968;33:473–484. doi: 10.1101/sqb.1968.033.01.055. [DOI] [PubMed] [Google Scholar]
  10. Horiuchi K., Zinder N. D. Origin and direction of synthesis of bacteriophage fl DNA. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2341–2345. doi: 10.1073/pnas.73.7.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hourcade D., Dressler D., Wolfson J. The nucleolus and the rolling circle. Cold Spring Harb Symp Quant Biol. 1974;38:537–550. doi: 10.1101/sqb.1974.038.01.058. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Marians K. J., Ikeda J. E., Schlagman S., Hurwitz J. Role of DNA gyrase in phiX replicative-form replication in vitro. Proc Natl Acad Sci U S A. 1977 May;74(5):1965–1968. doi: 10.1073/pnas.74.5.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meselson M., Stahl F. W. THE REPLICATION OF DNA IN ESCHERICHIA COLI. Proc Natl Acad Sci U S A. 1958 Jul 15;44(7):671–682. doi: 10.1073/pnas.44.7.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ogawa T., Tomizawa J. I., Fuke M. Replication of bacteriophage DNA, II. Structure of replicating DNA of phage lambda. Proc Natl Acad Sci U S A. 1968 Jul;60(3):861–865. doi: 10.1073/pnas.60.3.861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ray D. S. Replication of bacteriophage M13. II. The role of replicative forms in single-strand synthesis. J Mol Biol. 1969 Aug 14;43(3):631–643. doi: 10.1016/0022-2836(69)90364-7. [DOI] [PubMed] [Google Scholar]
  17. Schröder C. H., Kaerner H. C. Replication of bacteriophage phichi174 replicative form DNA in vivo. J Mol Biol. 1972 Nov 14;71(2):351–362. doi: 10.1016/0022-2836(72)90356-7. [DOI] [PubMed] [Google Scholar]
  18. Sinsheimer R. L. Bacteriophage phi-X174 and related viruses. Prog Nucleic Acid Res Mol Biol. 1968;8:115–169. [PubMed] [Google Scholar]
  19. Tessman E. S. Mutants of bacteriophage S13 blocked in infectious DNA synthesis. J Mol Biol. 1966 May;17(1):218–236. doi: 10.1016/s0022-2836(66)80104-3. [DOI] [PubMed] [Google Scholar]
  20. Vinograd J., Lebowitz J. Physical and topological properties of circular DNA. J Gen Physiol. 1966 Jul;49(6):103–125. doi: 10.1085/jgp.49.6.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Westmoreland B. C., Szybalski W., Ris H. Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy. Science. 1969 Mar 21;163(3873):1343–1348. doi: 10.1126/science.163.3873.1343. [DOI] [PubMed] [Google Scholar]
  22. Wolfson J., Dressler D., Magazin M. Bacteriophage T7 DNA replication: a linear replicating intermediate (gradient centrifugation-electron microscopy-E. coli-DNA partial denaturation). Proc Natl Acad Sci U S A. 1972 Feb;69(2):499–504. doi: 10.1073/pnas.69.2.499. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES