Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Nov 25;20(22):6075–6080. doi: 10.1093/nar/20.22.6075

DNA substrate specificity of DNA helicase E from calf thymus.

J J Turchi 1, R S Murante 1, R A Bambara 1
PMCID: PMC334475  PMID: 1334262

Abstract

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.

Full text

PDF
6075

Images in this article

6077Image
on p.6077
6079Image
on p.6079
6079Image
on p.6079

Selected References

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

  1. Araki H., Ropp P. A., Johnson A. L., Johnston L. H., Morrison A., Sugino A. DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J. 1992 Feb;11(2):733–740. doi: 10.1002/j.1460-2075.1992.tb05106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Coverley D., Kenny M. K., Munn M., Rupp W. D., Lane D. P., Wood R. D. Requirement for the replication protein SSB in human DNA excision repair. Nature. 1991 Feb 7;349(6309):538–541. doi: 10.1038/349538a0. [DOI] [PubMed] [Google Scholar]
  3. Dailey L., Caddle M. S., Heintz N., Heintz N. H. Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities. Mol Cell Biol. 1990 Dec;10(12):6225–6235. doi: 10.1128/mcb.10.12.6225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Lahue E. E., Matson S. W. Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction. J Biol Chem. 1988 Mar 5;263(7):3208–3215. [PubMed] [Google Scholar]
  5. Li X., Tan C. K., So A. G., Downey K. M. Purification and characterization of delta helicase from fetal calf thymus. Biochemistry. 1992 Apr 7;31(13):3507–3513. doi: 10.1021/bi00128a027. [DOI] [PubMed] [Google Scholar]
  6. Matson S. W. DNA helicases of Escherichia coli. Prog Nucleic Acid Res Mol Biol. 1991;40:289–326. doi: 10.1016/s0079-6603(08)60845-4. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. McHenry C., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Biol Chem. 1977 Sep 25;252(18):6478–6484. [PubMed] [Google Scholar]
  9. Mok M., Marians K. J. The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J Biol Chem. 1987 Dec 5;262(34):16644–16654. [PubMed] [Google Scholar]
  10. Mosbaugh D. W., Linn S. Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V. J Biol Chem. 1983 Jan 10;258(1):108–118. [PubMed] [Google Scholar]
  11. Mosbaugh D. W., Linn S. Gap-filling DNA synthesis by HeLa DNA polymerase alpha in an in vitro base excision DNA repair scheme. J Biol Chem. 1984 Aug 25;259(16):10247–10251. [PubMed] [Google Scholar]
  12. Nishida C., Reinhard P., Linn S. DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J Biol Chem. 1988 Jan 5;263(1):501–510. [PubMed] [Google Scholar]
  13. Runyon G. T., Bear D. G., Lohman T. M. Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6383–6387. doi: 10.1073/pnas.87.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sancar A., Rupp W. D. A novel repair enzyme: UVRABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell. 1983 May;33(1):249–260. doi: 10.1016/0092-8674(83)90354-9. [DOI] [PubMed] [Google Scholar]
  15. Seo Y. S., Lee S. H., Hurwitz J. Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity. J Biol Chem. 1991 Jul 15;266(20):13161–13170. [PubMed] [Google Scholar]
  16. Siegal G., Turchi J. J., Jessee C. B., Myers T. W., Bambara R. A. A novel DNA helicase from calf thymus. J Biol Chem. 1992 Jul 5;267(19):13629–13635. [PubMed] [Google Scholar]
  17. Thömmes P., Ferrari E., Jessberger R., Hübscher U. Four different DNA helicases from calf thymus. J Biol Chem. 1992 Mar 25;267(9):6063–6073. [PubMed] [Google Scholar]
  18. Thömmes P., Hübscher U. Eukaryotic DNA helicases. FEBS Lett. 1990 Aug 1;268(2):325–328. doi: 10.1016/0014-5793(90)81279-w. [DOI] [PubMed] [Google Scholar]
  19. Tuteja N., Rahman K., Tuteja R., Falaschi A. DNA helicase IV from HeLa cells. Nucleic Acids Res. 1991 Jul 11;19(13):3613–3618. doi: 10.1093/nar/19.13.3613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tuteja N., Tuteja R., Rahman K., Kang L. Y., Falaschi A. A DNA helicase from human cells. Nucleic Acids Res. 1990 Dec 11;18(23):6785–6792. doi: 10.1093/nar/18.23.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Zhang S. S., Grosse F. Purification and characterization of two DNA helicases from calf thymus nuclei. J Biol Chem. 1991 Oct 25;266(30):20483–20490. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES