Abstract
Replication fork helicases unwind DNA at a replication fork, providing polymerases with single-stranded DNA templates for replication. In bacteria, DnaB unwinds DNA at a replication fork, while in archaeal and eukaryotic organisms the Mcm proteins catalyze replication fork unwinding. Unwinding in archaea is catalyzed by a single Mcm protein that forms multimeric rings, whereas eukaryotic helicase activity is catalyzed by the heterohexameric Mcm2–7 complex acting in concert with Cdc45 and the GINS complex. A subcomplex of eukaryotic Mcm proteins, the Mcm4,6,7 complex, unwinds DNA in vitro, and studies of this assembly reveal insight into the mechanism of the eukaryotic Mcm helicase. Detailed methods for the investigation of replication fork helicase mechanism are described in this chapter. Described herein are methods for the design of DNA substrates for unwinding and branch migration studies, annealing DNA, purifying replication fork helicase proteins, and analyzing DNA unwinding activity.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Labib K., Tercero J. A., and Diffley J. F. X. (2000) Uninterrupted MCM2–7 function required for DNA replication fork progression. Science 288, 1643–1647.
Pacek M., Tutter A., Kubota Y., Takisawa H., and Walter J. (2006) Localization of MCM2–7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol. Cell 21, 581–587.
Calzada A., Hodgson B., Kanemaki M., Bueno A., and Labib K. (2005) Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev. 19, 1905–1919.
Moyer S., Lewis P., and Botchan M. (2006) Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA 103, 10236–10241.
Bochman M. and Schwacha A. (2008) The Mcm2-7 complex has in vitro helicase activity. Mol. Cell 31, 287–293.
Shechter D. F., Ying C. Y., and Gautier J. (2000) The intrinsic DNA helicase activity of Methanobacterium thermoautotrohicum delta H minichromosome maintenance protein. J. Biol. Chem. 275, 15049–15059.
Kelman Z., Lee J.-K., and Hurwitz J. (1999) The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity. Proc. Natl. Acad. Sci. USA 96, 14783–14788.
Chong J. P. J., Hayashi M. K., Simon M. N., Xu R.-M., and Stillman B. (2000) A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc. Natl. Acad. Sci. U.S.A. 97, 1530–1535.
Costa A., Pape T., van Heel M., Brick P., Patwardhan A., and Onesti S. (2006) Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity. Nucleic Acids Res. 34, 5829–5838.
Gomez-Llorente Y., Fletcher R., Chex X., Carazo X., and San Martin C. (2005) Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautrophicum. J. Biol. Chem. 280, 40909–40915.
Fletcher R., Shen J., Gomez-Llorente Y., Martin C. S., Carazo J. M., and Chen X. (2005) Double hexamer disruption and biochemical activities of Methanobacterium thermoautotrophicum MCM. J. Biol. Chem. 280, 42405–42410.
Ishimi Y. (1997) A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J. Biol. Chem. 272, 24508–24513.
Lee J.-K., and Hurwitz J. (2000) Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe. J. Biol. Chem. 275, 18871–18878.
You Z., Komamura Y., and Ishimi Y. (1999) Biochemical analysis of the intrinsic Mcm4-Mcm6-Mcm7 DNA helicase activity. Mol. Cell. Biol. 19, 8003–8015.
Kaplan D. L., Davey M. J., and O‘Donnell M. (2003) Mcm4,6,7 uses a ‘pump in ring’ mechanism to unwind DNA by steric exclusion and actively translocate along a duplex. J. Biol. Chem. 278, 49171–49182.
Lee J.-K., and Hurwitz J. (2001) Processive DNA helicase activity of the minichromosome maintenance proteins 4,6, and 7 complex requires forked DNA structures. Proc. Natl. Acad. Sci. USA 98, 54–59.
Kaplan D. L., and Steitz T. A. (1999) DnaB from Thermus aquaticus unwinds forked duplex DNA with an asymmetric tail length dependence. J. Biol. Chem. 274, 6889–6897.
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.
Kaplan D. L., and O’Donnell M. (2002) DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands. Mol. Cell 10, 647–657.
Kaplan D. L., and O’Donnell M. (2004) Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes. Mol. Cell 15, 453–465.
Kaplan D. and O’Donnell M. (2006) RuvA is a sliding collar that protects holliday junctions from unwinding while promoting branch migration. J. Mol. Biol. 355, 473–490.
Yuzhakov A., Turner J., and O’Donnell M. (1996) Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication. Cell 86, 877–886.
Davey M. J., Indiani C., and O’Donnell M. (2003) Reconstitution of the Mcm2–7p heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278, 4491–4499.
Acknowledgments
The authors thank Dr. Thomas A. Steitz and Dr. Mike O’Donnell for their support and encouragement.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kaplan, D.L., Bruck, I. (2009). Methods to Study How Replication Fork Helicases Unwind DNA. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-355-8_9
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-354-1
Online ISBN: 978-1-60327-355-8
eBook Packages: Springer Protocols