Skip to main content
NCBI 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
. 1981 Nov;78(11):6784–6788. doi: 10.1073/pnas.78.11.6784

Viable deletions of the M13 complementary strand origin

Myoung Hee Kim 1, Jane C Hines 1, Dan S Ray 1
PMCID: PMC349135  PMID: 6273888

Abstract

The single-stranded DNA of bacteriophage M13 is converted to a duplex replicative form by a mechanism involving RNA-primed initiation at a single unique site on the viral DNA. The DNA sequence that specifies the RNA primer is contained largely within one of two adjacent hairpin structures protected from DNase degradation by RNA polymerase. We have used in vitro techniques to construct a series of M13 mutants having deletions in the region of the complementary strand origin. Deletions of the duplex replicative form DNA range in size from 54 to 201 base pairs. The largest deletions remove both of the RNA polymerase-protected hairpins and the entire sequence specifying the primer RNA. Mutants lacking one or both hairpins form faint plaques, give reduced phage yields, and show a lag in phage production of >30 min. The rate of conversion of the single-stranded viral DNA to the parental replicative form is reduced both in vivo and in vitro. These results indicate that both the RNA polymerase-protected hairpins and the RNA primer-coding sequence are important, but not essential, for replication. Other sequences within the origin region, or possibly elsewhere in the genome, may play a role in complementary strand initiation in these mutant phages. The M13 viral strand is initiated by extension of the 3′ terminus generated by site-specific nicking of the viral strand of the replicative form DNA by the M13 gene II protein. This specific nicking site is retained in all of the M13 deletion mutants. Deletion end points do not extend into a 13-nucleotide sequence preceding the viral strand nicking site. We propose that a sequence including these 13 nucleotides is required for gene II protein action at this site.

Keywords: RNA primer, hairpins, gene II protein recognition sequence

Full text

PDF
6784

Images in this article

6785Image
on p.6785

Selected References

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

  1. Brutlag D., Schekman R., Kornberg A. A possible role for RNA polymerase in the initiation of M13 DNA synthesis. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2826–2829. doi: 10.1073/pnas.68.11.2826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cleary J. M., Ray D. S. Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4638–4642. doi: 10.1073/pnas.77.8.4638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Geider K., Beck E., Schaller H. An RNA transcribed from DNA at the origin of phage fd single strand to replicative form conversion. Proc Natl Acad Sci U S A. 1978 Feb;75(2):645–649. doi: 10.1073/pnas.75.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Geider K., Kornberg A. Conversion of the M13 viral single strand to the double-stranded replicative forms by purified proteins. J Biol Chem. 1974 Jul 10;249(13):3999–4005. [PubMed] [Google Scholar]
  5. Gray C. P., Sommer R., Polke C., Beck E., Schaller H. Structure of the orgin of DNA replication of bacteriophage fd. Proc Natl Acad Sci U S A. 1978 Jan;75(1):50–53. doi: 10.1073/pnas.75.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hines J. C., Ray D. S. Construction and characterization of new coliphage M13 cloning vectors. Gene. 1980 Nov;11(3-4):207–218. doi: 10.1016/0378-1119(80)90061-x. [DOI] [PubMed] [Google Scholar]
  7. Meyer T. F., Geider K., Kurz C., Schaller H. Cleavage site of bacteriophage fd gene II-protein in the origin of viral strand replication. Nature. 1979 Mar 22;278(5702):365–367. doi: 10.1038/278365a0. [DOI] [PubMed] [Google Scholar]
  8. Nomura N., Ray D. S. Expression of a DNA strand initiation sequence of ColE1 plasmid in a single-stranded DNA phage. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6566–6570. doi: 10.1073/pnas.77.11.6566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Osborne T. F., Schell R. E., Burch-Jaffe E., Berget S. J., Berk A. J. Mapping a eukaryotic promoter: a DNA sequence required for in vivo expression of adenovirus pre-early functions. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1381–1385. doi: 10.1073/pnas.78.3.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ray D. S., Kook K. Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13. Gene. 1978 Oct;4(2):109–119. doi: 10.1016/0378-1119(78)90024-0. [DOI] [PubMed] [Google Scholar]
  11. Ray D. S., Schekman R. W. Replication of bacteriophage M13. I. Sedimentation analysis of crude lysates of M13-infected bacteria. Biochim Biophys Acta. 1969 Apr 22;179(2):398–407. [PubMed] [Google Scholar]
  12. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Suggs S. V., Ray D. S. Nucleotide sequence of the origin for bacteriophage M13 DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):379–388. doi: 10.1101/sqb.1979.043.01.044. [DOI] [PubMed] [Google Scholar]
  14. Tabak H. F., Griffith J., Geider K., Schaller H., Kornberg A. Initiation of deoxyribonucleic acid synthesis. VII. A unique location of the gap in the M13 replicative duplex synthesized in vitro. J Biol Chem. 1974 May 25;249(10):3049–3054. [PubMed] [Google Scholar]
  15. Tamanoi F., Saito H., Richardson C. C. Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication. Proc Natl Acad Sci U S A. 1980 May;77(5):2656–2660. doi: 10.1073/pnas.77.5.2656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Van Den Hondel C. A., Pennings L., Schoenmakers J. G. Restriction-enzyme-cleavage maps of bacteriophage M13. Existence of an intergenic region on the M13 genome. Eur J Biochem. 1976 Sep;68(1):55–70. doi: 10.1111/j.1432-1033.1976.tb10764.x. [DOI] [PubMed] [Google Scholar]
  17. Vovis G. F., Horiuchi K., Zinder N. D. Endonuclease R-EcoRII restriction of bacteriophage f1 DNA in vitro: ordering of genes V and VII, location of an RNA promotor for gene VIII. J Virol. 1975 Sep;16(3):674–684. doi: 10.1128/jvi.16.3.674-684.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wickner W., Brutlag D., Schekman R., Kornberg A. RNA synthesis initiates in vitro conversion of M13 DNA to its replicative form. Proc Natl Acad Sci U S A. 1972 Apr;69(4):965–969. doi: 10.1073/pnas.69.4.965. [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