Abstract
DNA helicases utilize the energy of nucleotide hydrolysis to unwind the two annealed strands of the DNA helix and are involved in many aspects of DNA metabolism such as replication, recombination, and repair. Chromatin immunoprecipitation (ChIP) has been instrumental in determining the genomic targets of many DNA helicases and DNA helicase-containing complexes including the minichromosome maintenance (Mcm) proteins 2–7, the RecQ helicase Sgs1 as well as the Rvb1 and Rvb2 helicase-containing INO80 and SWR1 chromatin remodeling complexes. Here we describe a ChIP method that has been successfully used to map these proteins at chromosomal double-strand breaks and replication forks in the model organism Saccharomyces cerevisiae.
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
Gribun A., Cheung K. L., Huen J., Ortega J., and Houry W. A. (2008) Yeast Rvb1 and Rvb2 are ATP-dependent DNA helicases that form a heterohexameric complex. J. Mol. Biol. 376, 1320–1333.
Shen X., Mizuguchi G., Hamiche A., and Wu C. (2000) A chromatin remodelling complex involved in transcription and DNA processing. Nature 406, 541–544.
Mizuguchi G., Shen X., Landry J., Wu W. H., Sen S., and Wu C. (2004) ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303, 343–348.
Krogan N. J., Keogh M. C., Datta N., et al. (2003) A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. Mol. Cell. 12, 1565–1576.
Kobor M. S., Venkatasubrahmanyam S., Meneghini M. D., et al. (2004) A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2, E131.
van Attikum H., Fritsch O., Hohn B., and Gasser S. M. (2004) Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119, 777–788.
Morrison A. J., Highland J., Krogan N. J., et al. (2004) INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119, 767–775.
van Attikum H., Fritsch O., and Gasser S. M. (2007) Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks. EMBO J. 26, 4113–4125.
Shimada K., Oma Y., Schleker T., et al. (2008) Ino80 chromatin remodeling complex promotes recovery of stalled replication forks. Curr. Biol. 18, 566–575.
Papamichos-Chronakis M., and Peterson C. L. (2008) The Ino80 chromatin-remodeling enzyme regulates replisome function and stability. Nat. Struct. Mol. Biol. 15, 338–345.
Jonsson Z. O., Jha S., Wohlschlegel J. A., and Dutta A. (2004) Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex. Mol. Cell. 16, 465–477.
Biswas-Fiss E. E., Khopde S. M., and Biswas S. B. (2005) The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences. Biochemistry 44, 2916–2925.
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.
Maiorano D., Lutzmann M., and Mechali M. (2006) MCM proteins and DNA replication. Curr. Opin. Cell. Biol. 18, 130–136.
Aparicio O. M., Weinstein D. M., and Bell S. P. (1997) Components and dynamics of DNA replication complexes in Saccharomyces cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69.
Cobb J. A., Bjergbaek L., Shimada K., Frei C., and Gasser S. M. (2003) DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. EMBO J. 22, 4325–4336.
Bjergbaek L., Cobb J. A., Tsai-Pflugfelder M., and Gasser S. M. (2005) Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. EMBO J. 24, 405–417.
Cobb J. A., Schleker T., Rojas V., Bjergbaek L., Tercero J. A., and Gasser S. M. (2005) Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations. Genes Dev. 19, 3055–3069.
Lee S. E., Moore J. K., Holmes A., Umezu K., Kolodner R. D., and Haber J. E. (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94, 399–409.
Dubrana K., van Attikum H., Hediger F., and Gasser S. M. (2007) The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast. J. Cell. Sci. 120, 4209–4220.
van Werven F. J., and Timmers H. T. (2006) The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation. Nucleic Acids Res. 34, e33.
Acknowledgments
The authors would like to thank Professor Susan Gasser and members of the Gasser Laboratory for support during the development of these protocols. JC is an Alberta Heritage Foundation for Medical Research Scholar and work in the JC laboratory is funded by grants from the Canadian Institutes for Health Research # MOP-82736 and the Alberta Cancer Board # 23575. HvA is a Human Frontiers Science Program long-term fellow and work in HvA’s group is funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO).
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
Cobb, J., van Attikum, H. (2009). Mapping Genomic Targets of DNA Helicases by Chromatin Immunoprecipitation in Saccharomyces cerevisiae . 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-60327-355-8_8
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