Abstract
Fluorescent single-stranded DNA-binding proteins (SSB) that have a defined number of fluorophores per tetramer are invaluable tools to understand biochemical mechanism and biological function. Here, we describe the purification of fluorescent SSB chimeras with a unique number of fluorescent subunits incorporated per tetramer. We describe the use of these tetramers to enable clear visualization of SSB in vivo. Purified chimeras also facilitate single molecule studies (Liu et al., Protein Sci 20:1005–1020, 2011).
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
Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck JL (2008) SSB as an organizer/mobilizer of genome maintenance complexes. Crit Rev Biochem Mol Biol 43:289–318
Raghunathan S, Kozlov A, Lohman T, Waksman G (2000) Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat Struct Biol 7:648–652
Shereda RD, Bernstein DA, Keck JL (2007) A central role for SSB in Escherichia coli RecQ DNA helicase function. J Biol Chem 282:19247–19258
Buss JA, Kimura Y, Bianco PR (2008) RecG interacts directly with SSB: implications for stalled replication fork regression. Nucleic Acids Res 36:7029–7042
Suski C, Marians KJ (2008) Resolution of converging replication forks by RecQ and topoisomerase III. Mol Cell 30:779–789
Dillingham M, Tibbles K, Hunter J, Bell J, Kowalczykowski S, Webb M (2008) Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity. Biophys J 95:3330–3339
Roy R, Kozlov A, Lohman T, Ha T (2009) SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461:1092–1097
Reyes-Lamothe R, Possoz C, Danilova O, Sherratt DJ (2008) Independent positioning and action of Escherichia coli replisomes in live cells. Cell 133:90–102
Liu J, Choi M, Stanenas AG, Byrd AK, Raney KD, Cohan C, Bianco PR (2011) Novel, fluorescent, SSB protein chimeras with broad utility. Protein Sci 20:1005–1020
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bianco, P.R., Stanenas, A.J., Liu, J., Cohan, C.S. (2012). Fluorescent Single-Stranded DNA-Binding Proteins Enable In Vitro and In Vivo Studies. In: Keck, J. (eds) Single-Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 922. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-032-8_18
Download citation
DOI: https://doi.org/10.1007/978-1-62703-032-8_18
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-031-1
Online ISBN: 978-1-62703-032-8
eBook Packages: Springer Protocols