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. 1994 Jul 5;91(14):6644–6648. doi: 10.1073/pnas.91.14.6644

A fluorescence-based assay for monitoring helicase activity.

K D Raney 1, L C Sowers 1, D P Millar 1, S J Benkovic 1
PMCID: PMC44259  PMID: 8022830

Abstract

A continuous fluorescence-based assay is described for measuring helicase-mediated unwinding of duplex DNA. The assay utilizes an oligonucleotide substrate containing the fluorescent adenine analog, 2-aminopurine, at regular intervals. 2-Aminopurine forms a Watson-Crick-type base pair with thymine and does not distort normal B-form DNA. Fluorescence of the 2-aminopurines within this oligonucleotide is quenched 2-fold upon its hybridization to a complementary strand. Unwinding of this substrate by the T4 dda helicase restores the fluorescence of the 2-aminopurines and is easily followed using stopped-flow or steady-state fluorescence spectroscopy. The flourescence-based assay provides rate data comparable to that obtained from conventional discontinuous assays using labeled substrates and additionally furnishes a means for following a single turnover. This assay should prove useful for defining the mechanism by which helicases unwind duplex DNA.

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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. Bloom L. B., Otto M. R., Beechem J. M., Goodman M. F. Influence of 5'-nearest neighbors on the insertion kinetics of the fluorescent nucleotide analog 2-aminopurine by Klenow fragment. Biochemistry. 1993 Oct 19;32(41):11247–11258. doi: 10.1021/bi00092a039. [DOI] [PubMed] [Google Scholar]
  3. Capson T. L., Peliska J. A., Kaboord B. F., Frey M. W., Lively C., Dahlberg M., Benkovic S. J. Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry. 1992 Nov 17;31(45):10984–10994. doi: 10.1021/bi00160a007. [DOI] [PubMed] [Google Scholar]
  4. Geiselmann J., Wang Y., Seifried S. E., von Hippel P. H. A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7754–7758. doi: 10.1073/pnas.90.16.7754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Geiselmann J., Yager T. D., Gill S. C., Calmettes P., von Hippel P. H. Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer. Biochemistry. 1992 Jan 14;31(1):111–121. doi: 10.1021/bi00116a017. [DOI] [PubMed] [Google Scholar]
  6. Geiselmann J., von Hippel P. H. Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP. Protein Sci. 1992 Jul;1(7):850–860. doi: 10.1002/pro.5560010703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guest C. R., Hochstrasser R. A., Sowers L. C., Millar D. P. Dynamics of mismatched base pairs in DNA. Biochemistry. 1991 Apr 2;30(13):3271–3279. doi: 10.1021/bi00227a015. [DOI] [PubMed] [Google Scholar]
  8. Hacker K. J., Alberts B. M. Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase. J Biol Chem. 1992 Oct 15;267(29):20674–20681. [PubMed] [Google Scholar]
  9. Lohman T. M. Helicase-catalyzed DNA unwinding. J Biol Chem. 1993 Feb 5;268(4):2269–2272. [PubMed] [Google Scholar]
  10. Maine I. P., Sun D., Hurley L. H., Kodadek T. The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA. Biochemistry. 1992 Apr 28;31(16):3968–3975. doi: 10.1021/bi00131a012. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Nordlund T. M., Andersson S., Nilsson L., Rigler R., Gräslund A., McLaughlin L. W. Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies. Biochemistry. 1989 Nov 14;28(23):9095–9103. doi: 10.1021/bi00449a021. [DOI] [PubMed] [Google Scholar]
  13. Roman L. J., Kowalczykowski S. C. Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry. 1989 Apr 4;28(7):2863–2873. doi: 10.1021/bi00433a018. [DOI] [PubMed] [Google Scholar]
  14. SenGupta D. J., Borowiec J. A. Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen. Science. 1992 Jun 19;256(5064):1656–1661. doi: 10.1126/science.256.5064.1656. [DOI] [PubMed] [Google Scholar]
  15. Sowers L. C., Fazakerley G. V., Eritja R., Kaplan B. E., Goodman M. F. Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5434–5438. doi: 10.1073/pnas.83.15.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]

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