Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Sep;10(9):2689–2694. doi: 10.1002/j.1460-2075.1991.tb07812.x

Endonuclease activity of Escherichia coli DNA helicase I directed against the transfer origin of the F factor.

U Reygers 1, R Wessel 1, H Müller 1, H Hoffmann-Berling 1
PMCID: PMC452971  PMID: 1651233

Abstract

DNA helicase I, the traI gene product of the Escherichia coli F factor, was shown to be associated with endonuclease activity specific for the transfer origin of the F plasmid, oriT. In the presence of Mg2+, the purified enzyme forms a complex, stable in the presence of sodium dodecylsulfate (SDS) with a negatively superhelical chimeric plasmid containing oriT. The enzyme nicks and, after this, apparently binds to the 5' nick terminus when this complex is heated in the presence of SDS and/or EDTA or treated with proteinase K. Dideoxy sequencing locates the nick site in the F DNA strand transferred during bacterial conjugation after nucleotide 138 clockwise of the mid-point of the BglII site at 66.7 kb of the F genetic map. A sequencing stop after nucleotide 137 of this strand (where oriT-nicking seems to occur in vivo) is possibly an artefact caused by helicase I protein attached to the 5' terminal nucleotide. Deletion in the amino-terminal part of the traI polypeptide abolishes the oriT-nicking activity while leaving the strand-separating activity intact. These results confirm the prediction from genetic studies that helicase I is bifunctional with site-specific endonuclease and strand-separating activities.

Full text

PDF

Images in this article

2690Image
on p.2690
2690Image
on p.2690
2690Image
on p.2690
2691Image
on p.2691
2692Image
on p.2692
2693Image
on p.2693

Selected References

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

  1. Abdel-Monem M., Taucher-Scholz G., Klinkert M. Q. Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4659–4663. doi: 10.1073/pnas.80.15.4659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benz I., Müller H. Escherichia coli DNA helicase I. Characterization of the protein and of its DNA-binding properties. Eur J Biochem. 1990 Apr 30;189(2):267–276. doi: 10.1111/j.1432-1033.1990.tb15486.x. [DOI] [PubMed] [Google Scholar]
  3. Bradshaw H. D., Jr, Traxler B. A., Minkley E. G., Jr, Nester E. W., Gordon M. P. Nucleotide sequence of the traI (helicase I) gene from the sex factor F. J Bacteriol. 1990 Jul;172(7):4127–4131. doi: 10.1128/jb.172.7.4127-4131.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dodson M., Dean F. B., Bullock P., Echols H., Hurwitz J. Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science. 1987 Nov 13;238(4829):964–967. doi: 10.1126/science.2823389. [DOI] [PubMed] [Google Scholar]
  6. Everett R., Willetts N. Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F. J Mol Biol. 1980 Jan 15;136(2):129–150. doi: 10.1016/0022-2836(80)90309-5. [DOI] [PubMed] [Google Scholar]
  7. Finlay B. B., Frost L. S., Paranchych W. Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1. J Bacteriol. 1986 Oct;168(1):132–139. doi: 10.1128/jb.168.1.132-139.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ippen-Ihler K. A., Minkley E. G., Jr The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet. 1986;20:593–624. doi: 10.1146/annurev.ge.20.120186.003113. [DOI] [PubMed] [Google Scholar]
  9. Kowalski D., Sanford J. P. Action of mung bean nuclease on supercoiled PM2 DNA. J Biol Chem. 1982 Jul 10;257(13):7820–7825. [PubMed] [Google Scholar]
  10. Lahue E. E., Matson S. W. Purified Escherichia coli F-factor TraY protein binds oriT. J Bacteriol. 1990 Mar;172(3):1385–1391. doi: 10.1128/jb.172.3.1385-1391.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lanka E., Scherzinger E., Günther E., Schuster H. A DNA primase specified by I-like plasmids. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3632–3636. doi: 10.1073/pnas.76.8.3632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Liu L. F., Wang J. C. Interaction between DNA and Escherichia coli DNA topoisomerase I. Formation of complexes between the protein and superhelical and nonsuperhelical duplex DNAs. J Biol Chem. 1979 Nov 10;254(21):11082–11088. [PubMed] [Google Scholar]
  13. Manning P. A., Kusecek B., Morelli G., Fisseau C., Achtman M. Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2. J Bacteriol. 1982 Apr;150(1):76–88. doi: 10.1128/jb.150.1.76-88.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Scheffner M., Wessel R., Stahl H. Sequence independent duplex DNA opening reaction catalysed by SV40 large tumor antigen. Nucleic Acids Res. 1989 Jan 11;17(1):93–106. doi: 10.1093/nar/17.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Thompson R., Achtman M. The control region of the F sex factor DNA transfer cistrons: restriction mapping and DNA cloning. Mol Gen Genet. 1978 Oct 24;165(3):295–304. doi: 10.1007/BF00332530. [DOI] [PubMed] [Google Scholar]
  18. Thompson R., Taylor L., Kelly K., Everett R., Willetts N. The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks. EMBO J. 1984 May;3(5):1175–1180. doi: 10.1002/j.1460-2075.1984.tb01947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Thompson T. L., Centola M. B., Deonier R. C. Location of the nick at oriT of the F plasmid. J Mol Biol. 1989 Jun 5;207(3):505–512. doi: 10.1016/0022-2836(89)90460-9. [DOI] [PubMed] [Google Scholar]
  20. Traxler B. A., Minkley E. G., Jr Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein. J Mol Biol. 1988 Nov 5;204(1):205–209. doi: 10.1016/0022-2836(88)90609-2. [DOI] [PubMed] [Google Scholar]
  21. Traxler B. A., Minkley E. G., Jr Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport. J Bacteriol. 1987 Jul;169(7):3251–3259. doi: 10.1128/jb.169.7.3251-3259.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wessel R., Müller H., Hoffmann-Berling H. Electron microscopy of DNA.helicase-I complexes in the act of strand separation. Eur J Biochem. 1990 Apr 30;189(2):277–285. doi: 10.1111/j.1432-1033.1990.tb15487.x. [DOI] [PubMed] [Google Scholar]
  23. Willetts N., Maule J. Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages. Mol Gen Genet. 1979 Feb 1;169(3):325–336. doi: 10.1007/BF00382278. [DOI] [PubMed] [Google Scholar]
  24. Willetts N., Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. doi: 10.1128/mr.48.1.24-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Willetts N., Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. doi: 10.1128/mr.48.1.24-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van der Ende A., Langeveld S. A., Teertstra R., van Arkel G. A., Weisbeek P. J. Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication. Nucleic Acids Res. 1981 May 11;9(9):2037–2053. doi: 10.1093/nar/9.9.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES