Abstract
Efficient replication and repair of the genome requires a multitude of protein–DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein–DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bochman ML (2014) Roles of DNA helicases in the maintenance of genome integrity. Mol Cell Oncol 1(3):e963429. https://doi.org/10.4161/23723548.2014.963429
Brosh RM Jr (2016) Special methods collection on DNA helicases. Methods 108:1–3. https://doi.org/10.1016/j.ymeth.2016.08.009
Hodeib S, Raj S, Manosas M, Zhang W, Bagchi D, Ducos B, Fiorini F, Kanaan J, Le Hir H, Allemand JF, Bensimon D, Croquette V (2017) A mechanistic study of helicases with magnetic traps. Protein Sci 26(7):1314–1336. https://doi.org/10.1002/pro.3187
Cejka P, Kowalczykowski SC (2010) The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds Holliday junctions. J Biol Chem 285(11):8290–8301. https://doi.org/10.1074/jbc.M109.083196
Rogers CM, Wang JC, Noguchi H, Imasaki T, Takagi Y, Bochman ML (2017) Yeast Hrq1 shares structural and functional homology with the disease-linked human RecQ4 helicase. Nucleic Acids Res 45(9):5217–5230. https://doi.org/10.1093/nar/gkx151
Bhargava R, Onyango DO, Stark JM (2016) Regulation of single-strand annealing and its role in genome maintenance. Trends Genet 32(9):566–575. https://doi.org/10.1016/j.tig.2016.06.007
Macris MA, Krejci L, Bussen W, Shimamoto A, Sung P (2006) Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome. DNA Repair 5(2):172–180. https://doi.org/10.1016/j.dnarep.2005.09.005
Keller H, Kiosze K, Sachsenweger J, Haumann S, Ohlenschlager O, Nuutinen T, Syvaoja JE, Gorlach M, Grosse F, Pospiech H (2014) The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding. Nucleic Acids Res 42(20):12614–12627. https://doi.org/10.1093/nar/gku993
Daley JM, Kwon Y, Niu H, Sung P (2013) Investigations of homologous recombination pathways and their regulation. Yale J Biol Med 86(4):453–461
Kanter DM, Kaplan DL (2011) Sld2 binds to origin single-stranded DNA and stimulates DNA annealing. Nucleic Acids Res 39(7):2580–2592. https://doi.org/10.1093/nar/gkq1222
Rogers CM, Bochman ML (2017) Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA. Biochem Biophys Res Commun 486(4):1116–1121. https://doi.org/10.1016/j.bbrc.2017.04.003
Acknowledgments
Funding: This work was supported by the College of Arts and Sciences, Indiana University, and Indiana University Collaborative Research Grant fund of the Office of the Vice President for Research, a Collaboration in Translational Research Pilot Grant from the Indiana Clinical and Translational Sciences Institute, and the American Cancer Society [RSG-16-180-01-DMC].
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Rogers, C.M., Sausen, C.W., Bochman, M.L. (2019). Gel-Based Assays for Measuring DNA Unwinding, Annealing, and Strand Exchange. In: Balakrishnan, L., Stewart, J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9500-4_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9500-4_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9499-1
Online ISBN: 978-1-4939-9500-4
eBook Packages: Springer Protocols