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
. 1996 Sep 17;93(19):10051–10056. doi: 10.1073/pnas.93.19.10051

ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: implications for ATP driven helicase translocation.

I Wong 1, T M Lohman 1
PMCID: PMC38334  PMID: 8816749

Abstract

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

Full text

PDF
10051

Images in this article

10053Fig. 1
on p.10053

Selected References

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

  1. Amaratunga M., Lohman T. M. Escherichia coli rep helicase unwinds DNA by an active mechanism. Biochemistry. 1993 Jul 13;32(27):6815–6820. doi: 10.1021/bi00078a003. [DOI] [PubMed] [Google Scholar]
  2. Bjornson K. P., Moore K. J., Lohman T. M. Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase. Biochemistry. 1996 Feb 20;35(7):2268–2282. doi: 10.1021/bi9522763. [DOI] [PubMed] [Google Scholar]
  3. Brown W. C., Romano L. J. Benzo[a]pyrene-DNA adducts inhibit translocation by the gene 4 protein of bacteriophage T7. J Biol Chem. 1989 Apr 25;264(12):6748–6754. [PubMed] [Google Scholar]
  4. Chao K. L., Lohman T. M. DNA-induced dimerization of the Escherichia coli Rep helicase. J Mol Biol. 1991 Oct 20;221(4):1165–1181. doi: 10.1016/0022-2836(91)90926-w. [DOI] [PubMed] [Google Scholar]
  5. Colasanti J., Denhardt D. T. The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels. Mol Gen Genet. 1987 Sep;209(2):382–390. doi: 10.1007/BF00329669. [DOI] [PubMed] [Google Scholar]
  6. Friedberg E. C. Xeroderma pigmentosum, Cockayne's syndrome, helicases, and DNA repair: what's the relationship? Cell. 1992 Dec 11;71(6):887–889. doi: 10.1016/0092-8674(92)90384-o. [DOI] [PubMed] [Google Scholar]
  7. Gilbert S. P., Webb M. R., Brune M., Johnson K. A. Pathway of processive ATP hydrolysis by kinesin. Nature. 1995 Feb 23;373(6516):671–676. doi: 10.1038/373671a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hackney D. D. Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6865–6869. doi: 10.1073/pnas.91.15.6865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lohman T. M., Bjornson K. P. Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem. 1996;65:169–214. doi: 10.1146/annurev.bi.65.070196.001125. [DOI] [PubMed] [Google Scholar]
  10. Lohman T. M., Bujalowski W. Negative cooperativity within individual tetramers of Escherichia coli single strand binding protein is responsible for the transition between the (SSB)35 and (SSB)56 DNA binding modes. Biochemistry. 1988 Apr 5;27(7):2260–2265. doi: 10.1021/bi00407a002. [DOI] [PubMed] [Google Scholar]
  11. Lohman T. M., Chao K., Green J. M., Sage S., Runyon G. T. Large-scale purification and characterization of the Escherichia coli rep gene product. J Biol Chem. 1989 Jun 15;264(17):10139–10147. [PubMed] [Google Scholar]
  12. Lohman T. M. Escherichia coli DNA helicases: mechanisms of DNA unwinding. Mol Microbiol. 1992 Jan;6(1):5–14. doi: 10.1111/j.1365-2958.1992.tb00831.x. [DOI] [PubMed] [Google Scholar]
  13. Lohman T. M. Helicase-catalyzed DNA unwinding. J Biol Chem. 1993 Feb 5;268(4):2269–2272. [PubMed] [Google Scholar]
  14. Matson S. W., Kaiser-Rogers K. A. DNA helicases. Annu Rev Biochem. 1990;59:289–329. doi: 10.1146/annurev.bi.59.070190.001445. [DOI] [PubMed] [Google Scholar]
  15. Modrich P. Mismatch repair, genetic stability, and cancer. Science. 1994 Dec 23;266(5193):1959–1960. doi: 10.1126/science.7801122. [DOI] [PubMed] [Google Scholar]
  16. Moore K. J., Lohman T. M. Helicase-catalyzed DNA unwinding: energy coupling by DNA motor proteins. Biophys J. 1995 Apr;68(4 Suppl):180S–185S. [PMC free article] [PubMed] [Google Scholar]
  17. Moore K. J., Lohman T. M. Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogues. Biochemistry. 1994 Dec 6;33(48):14550–14564. doi: 10.1021/bi00252a023. [DOI] [PubMed] [Google Scholar]
  18. Raney K. D., Benkovic S. J. Bacteriophage T4 Dda helicase translocates in a unidirectional fashion on single-stranded DNA. J Biol Chem. 1995 Sep 22;270(38):22236–22242. doi: 10.1074/jbc.270.38.22236. [DOI] [PubMed] [Google Scholar]
  19. Sancar A. Mechanisms of DNA excision repair. Science. 1994 Dec 23;266(5193):1954–1956. doi: 10.1126/science.7801120. [DOI] [PubMed] [Google Scholar]
  20. Wong I., Amaratunga M., Lohman T. M. Heterodimer formation between Escherichia coli Rep and UvrD proteins. J Biol Chem. 1993 Sep 25;268(27):20386–20391. [PubMed] [Google Scholar]
  21. Wong I., Chao K. L., Bujalowski W., Lohman T. M. DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA. J Biol Chem. 1992 Apr 15;267(11):7596–7610. [PubMed] [Google Scholar]
  22. Wong I., Lohman T. M. Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding. Science. 1992 Apr 17;256(5055):350–355. doi: 10.1126/science.256.5055.350. [DOI] [PubMed] [Google Scholar]
  23. Wong I., Moore K. J., Bjornson K. P., Hsieh J., Lohman T. M. ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states. Biochemistry. 1996 May 7;35(18):5726–5734. doi: 10.1021/bi952959i. [DOI] [PubMed] [Google Scholar]
  24. Young M. C., Schultz D. E., Ring D., von Hippel P. H. Kinetic parameters of the translocation of bacteriophage T4 gene 41 protein helicase on single-stranded DNA. J Mol Biol. 1994 Feb 4;235(5):1447–1458. doi: 10.1006/jmbi.1994.1100. [DOI] [PubMed] [Google Scholar]
  25. Zimmerle C. T., Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J. 1989 Mar 1;258(2):381–387. doi: 10.1042/bj2580381. [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