Abstract
Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5′-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Singleton, M. R. and Wigley, D. B. (2003) Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes. EMBO J. 22, 4579–4583.
Caruthers, J. M. and McKay, D. B. (2002) Helicase structure and mechanism. Curr. Opin. Struct. Biol. 12, 123–133.
Delagoutte, E. and von Hippel, P. H. (2002) Helicase mechanisms and the coupling of helicases within macromolecular machines. Part I: Structures and properties of isolated helicases. Q. Rev. Biophys. 35, 431–478.
Hall, M. C. and Matson, S. W. (1999) Helicase motifs: the engine that powers DNA unwinding. Mol. Microbiol. 34, 867–877.
Patel, S. S. and Picha, K. M. (2000) Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697.
Bachrati, C. Z. and Hickson, I. D. (2003) RecQ helicases: suppressors of tumorigenesis and premature ageing. Biochem. J. 374, 577–606.
Brosh, R. M. Jr. and Bohr, V. A. (2002) Roles of the Werner syndrome protein in pathways required for maintenance of genome stability. Exp. Gerontol. 37, 491–506.
Opresko, P. L., Cheng, W. H., and Bohr, V. A. (2004) At the junction of RecQ helicase biochemistry and human disease. J. Biol. Chem. 279, 18,099–18,102. E-Pub 15023996.
Matson, S. W. (1986) Escherichia coli helicase II (urvD gene product) translocates unidirectionally in a 3′ to 5′ direction. J. Biol. Chem. 261, 10,169–10,175.
Matson, S. W., Bean, D. W., and George, J. W. (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioessays 16, 13–22.
Dillingham, M. S., Wigley, D. B., and Webb, M. R. (2002) Direct measurement of single-stranded DNA translocation by PcrA helicase using the fluorescent base analogue 2-aminopurine. Biochemistry 41, 643–651.
Dillingham, M. S., Wigley, D. B., and Webb, M. R. (2000) Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed. Biochemistry 39, 205–212.
Ahnert, P. and Patel, S. S. (1997) Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5′-and 3′-tails of the forked DNA substrate. J. Biol. Chem. 272, 32,267–32,273.
Brosh, R. M. Jr., Waheed, J., and Sommers, J. A. (2002) Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase. J. Biol. Chem. 277, 23,236–23,245.
Kaplan, D. L. (2000) The 3′-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase. J. Mol. Biol. 301, 285–299.
Sun, H., Karow, J. K., Hickson, I. D., and Maizels, N. (1998) The Bloom’s syndrome helicase unwinds G4 DNA. J. Biol. Chem. 273, 27,587–27,592.
Morris, P. D. and Raney, K. D. (1999) DNA helicases displace streptavidin from biotin-labeled oligonucleotides. Biochemistry 38, 5164–5171.
Morris, P. D., Byrd, A. K., Tackett, A. J., et al. (2002) Hepatitis C virus NS3 and Simian virus 40 T antigen helicases displace streptavidin from 5′-biotinylated oligonucleotides but not from 3′-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA. Biochemistry 41, 2372–2378.
Brosh, R. M. Jr., Orren, D. K., Nehlin, J. O., et al. (1999) Functional and physical interaction between WRN helicase and human Replication Protein A. J. Biol. Chem. 274, 18,341–18,350.
Shen, J. C., Gray, M. D., Oshima, J., and Loeb, L. A. (1998) Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by Replication Protein A. Nucleic. Acids. Res. 26, 2879–2885.
Brosh, R. M. Jr., Li, J. L., Kenny, M. K., et al. (2000) Replication Protein A physically interacts with the Bloom’s syndrome protein and stimulates its helicase activity. J. Biol. Chem. 275, 23,500–23,508.
Cui, S., Klima, R., Ochem, A., Arosio, D., Falaschi, A., and Vindigni, A. (2003) Characterization of the DNA-unwinding activity of human RECQ1, a helicase specifically stimulated by human Replication Protein A. J. Biol. Chem. 278, 1424–1432.
Kikuma, T., Ohtsu, M., Utsugi, T., et al. (2004) Dbp9p, a member of DEAD box protein family, has DNA helicase activity. J. Biol. Chem. E-Pub. 15028736.
Brosh, R. M. Jr., Majumdar, A., Desai, S., Hickson, I. D., Bohr, V. A., and Seidman, M. M. (2001) Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases. J. Biol. Chem. 276, 3024–3030.
Villani, G. and Tanguy, L. G. (2000) Interactions of DNA helicases with damaged DNA: possible biological consequences. J. Biol. Chem. 275, 33,185–33,188.
Naegeli, H., Bardwell, L., and Friedberg, E. C. (1993) Inhibition of Rad3 DNA helicase activity by DNA adducts and abasic sites: implications for the role of a DNA helicase in damage-specific incision of DNA. Biochemistry 32, 613–621.
Driscoll, H. C., Matson, S. W., Sayer, J. M., Kroth, H., Jerina, D. M., and Brosh, R. M. Jr. (2003) Inhibition of Werner syndrome helicase activity by benzo[c]-phenanthrene diol epoxide dA adducts in DNA is both strand-and stereoisomer-dependent. J. Biol. Chem. 278, 41,126–41,135.
Harmon, F. G. and Kowalczykowski, S. C. (2001) Biochemical characterization of the DNA helicase activity of the Escherichia coli RecQ helicase. J. Biol. Chem. 276, 232–243.
Bjornson, K. P., Amaratunga, M., Moore, K. J., and Lohman, T. M. (1994) Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluorescence energy transfer. Biochemistry 33, 14,306–14,316.
Ali, J. A., Maluf, N. K., and Lohman, T. M. (1999) An oligomeric form of E. coli UvrD is required for optimal helicase activity. J. Mol. Biol. 293, 815–834.
Lucius, A. L., Vindigni, A., Gregorian, R., et al. (2002) DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies. J. Mol. Biol. 324, 409–428.
Nanduri, B., Byrd, A. K., Eoff, R. L., Tackett, A. J., and Raney, K. D. (2002) Presteady-state DNA unwinding by bacteriophage T4 Dda helicase reveals a monomeric molecular motor. Proc. Natl. Acad. Sci. USA 99, 14,722–14,727.
Mohaghegh, P., Karow, J. K., Brosh, R. M. Jr., Bohr, V. A., and Hickson, I. D. (2001) The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases. Nucleic. Acids. Res. 29, 2843–2849.
Sharma, S., Otterlei, M., Sommers, J. A., et al. (2004) WRN Helicase and FEN-1 form a complex upon replication arrest and together process branch-migrating DNA structures associated with the replication fork. Mol. Biol. Cell 15, 734–750.
Acknowledgments
Because of page length limitations, we were not able to reference a number of papers in the literature from laboratories that developed various biochemical techniques for the study of DNA helicases. We wish to thank our colleagues in The Laboratory of Molecular Gerontology, National Institute on Aging, NIH for helpful discussion.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Brosh, R.M., Sharma, S. (2006). Biochemical Assays for the Characterization of DNA Helicases. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:397
Download citation
DOI: https://doi.org/10.1385/1-59259-973-7:397
Publisher Name: Humana Press
Print ISBN: 978-1-58829-513-2
Online ISBN: 978-1-59259-973-8
eBook Packages: Springer Protocols