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. 1994 Jun 11;22(11):2065–2070. doi: 10.1093/nar/22.11.2065

Phage P4 DNA replication in vitro.

R Díaz Orejas 1, G Ziegelin 1, R Lurz 1, E Lanka 1
PMCID: PMC308122  PMID: 8029013

Abstract

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.

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Selected References

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

  1. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowden D. W., Twersky R. S., Calendar R. Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis. J Bacteriol. 1975 Oct;124(1):167–175. doi: 10.1128/jb.124.1.167-175.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Diaz R., Staudenbauer W. L. Replication of the broad host range plasmid RSF1010 in cell-free extracts of Escherichia coli and Pseudomonas aeruginosa. Nucleic Acids Res. 1982 Aug 11;10(15):4687–4702. doi: 10.1093/nar/10.15.4687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Flensburg J., Calendar R. Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region. J Mol Biol. 1987 May 20;195(2):439–445. doi: 10.1016/0022-2836(87)90664-4. [DOI] [PubMed] [Google Scholar]
  5. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  6. Giraldo-Suárez R., Fernández-Tresguerres E., Díaz-Orejas R., Malki A., Kohiyama M. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication. Nucleic Acids Res. 1993 Nov 25;21(23):5495–5499. doi: 10.1093/nar/21.23.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goetz G. S., Dean F. B., Hurwitz J., Matson S. W. The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity. J Biol Chem. 1988 Jan 5;263(1):383–392. [PubMed] [Google Scholar]
  8. Gutiérrez C., Sogo J. M., Salas M. Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro. J Mol Biol. 1991 Dec 20;222(4):983–994. doi: 10.1016/0022-2836(91)90589-x. [DOI] [PubMed] [Google Scholar]
  9. Halling C., Calendar R., Christie G. E., Dale E. C., Dehò G., Finkel S., Flensburg J., Ghisotti D., Kahn M. L., Lane K. B. DNA sequence of satellite bacteriophage P4. Nucleic Acids Res. 1990 Mar 25;18(6):1649–1649. doi: 10.1093/nar/18.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haring V., Scholz P., Scherzinger E., Frey J., Derbyshire K., Hatfull G., Willetts N. S., Bagdasarian M. Protein RepC is involved in copy number control of the broad host range plasmid RSF1010. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6090–6094. doi: 10.1073/pnas.82.18.6090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaguni J. M., Fuller R. S., Kornberg A. Enzymatic replication of E. coli chromosomal origin is bidirectional. Nature. 1982 Apr 15;296(5858):623–627. doi: 10.1038/296623a0. [DOI] [PubMed] [Google Scholar]
  12. Krevolin M. D., Calendar R. The replication of bacteriophage P4 DNA in vitro. Partial purification of the P4 alpha gene product. J Mol Biol. 1985 Apr 20;182(4):509–517. doi: 10.1016/0022-2836(85)90237-2. [DOI] [PubMed] [Google Scholar]
  13. Krevolin M. D., Inman R. B., Roof D., Kahn M., Calendar R. Bacteriophage P4 DNA replication. Location of the P4 origin. J Mol Biol. 1985 Apr 20;182(4):519–527. doi: 10.1016/0022-2836(85)90238-4. [DOI] [PubMed] [Google Scholar]
  14. Lindqvist B. H., Dehò G., Calendar R. Mechanisms of genome propagation and helper exploitation by satellite phage P4. Microbiol Rev. 1993 Sep;57(3):683–702. doi: 10.1128/mr.57.3.683-702.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindqvist B. H., Six E. W. Replication of bacteriophage P4 DNA in a nonlysogenic host. Virology. 1971 Jan;43(1):1–7. doi: 10.1016/0042-6822(71)90218-2. [DOI] [PubMed] [Google Scholar]
  16. Mastrangelo I. A., Hough P. V., Wall J. S., Dodson M., Dean F. B., Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature. 1989 Apr 20;338(6217):658–662. doi: 10.1038/338658a0. [DOI] [PubMed] [Google Scholar]
  17. Molin S., Nordström K. Control of plasmid R1 replication: functions involved in replication, copy number control, incompatibility, and switch-off of replication. J Bacteriol. 1980 Jan;141(1):111–120. doi: 10.1128/jb.141.1.111-120.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ortega S., Lanka E., Diaz R. The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA. Nucleic Acids Res. 1986 Jun 25;14(12):4865–4879. doi: 10.1093/nar/14.12.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scherzinger E., Haring V., Lurz R., Otto S. Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins. Nucleic Acids Res. 1991 Mar 25;19(6):1203–1211. doi: 10.1093/nar/19.6.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schuster H., Mikolajczyk M., Rohrschneider J., Geschke B. phiX174 DNA-dependent DNA synthesis in vitro: requirement for P1 ban protein in dnaB mutant extracts of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3907–3911. doi: 10.1073/pnas.72.10.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Seufert W., Dobrinski B., Lurz R., Messer W. Functionality of the dnaA protein binding site in DNA replication is orientation-dependent. J Biol Chem. 1988 Feb 25;263(6):2719–2723. [PubMed] [Google Scholar]
  22. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  23. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Strack B., Lessl M., Calendar R., Lanka E. A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. J Biol Chem. 1992 Jun 25;267(18):13062–13072. [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wickner S. H. Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2690–2694. doi: 10.1073/pnas.87.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yochem J., Uchida H., Sunshine M., Saito H., Georgopoulos C. P., Feiss M. Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication. Mol Gen Genet. 1978 Aug 4;164(1):9–14. doi: 10.1007/BF00267593. [DOI] [PubMed] [Google Scholar]
  30. Ziegelin G., Scherzinger E., Lurz R., Lanka E. Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities. EMBO J. 1993 Sep;12(9):3703–3708. doi: 10.1002/j.1460-2075.1993.tb06045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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