Abstract
Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.
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
Lecointe F, Serena C, Velten M, Costes A, McGovern S, Meile JC, Errington J, Ehrlich SD, Noirot P, Polard P (2007) Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks. EMBO J 26(19):4239–4251
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(5):289–318. https://doi.org/10.1080/10409230802341296
Sun Z, Hashemi M, Warren G, Bianco PR, Lyubchenko YL (2018) Dynamics of the interaction of RecG protein with stalled replication forks. Biochemistry 57(13):1967–1976. https://doi.org/10.1021/acs.biochem.7b01235
Bianco PR, Lyubchenko YL (2017) SSB and the RecG DNA helicase: an intimate association to rescue a stalled replication fork. Protein Sci 26(4):638–649. https://doi.org/10.1002/pro.3114
Bianco PR (2017) The tale of SSB. Prog Biophys Mol Biol 127:111–118. https://doi.org/10.1016/j.pbiomolbio.2016.11.001
Manosas M, Perumal SK, Bianco PR, Ritort F, Benkovic SJ, Croquette V (2013) RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue. Nat Commun 4:2368. https://doi.org/10.1038/ncomms3368
Buss J, Kimura Y, Bianco P (2008) RecG interacts directly with SSB: implications for stalled replication fork regression. Nucleic Acids Res 36(22):7029–7042
Yu C, Tan HY, Choi M, Stanenas AJ, Byrd AK, K DR, Cohan CS, Bianco PR (2016) SSB binds to the RecG and PriA helicases in vivo in the absence of DNA. Genes Cells 21(2):163–184. https://doi.org/10.1111/gtc.12334
Slocum SL, Buss JA, Kimura Y, Bianco PR (2007) Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA. J Mol Biol 367(3):647–664. https://doi.org/10.1016/j.jmb.2007.01.007
Handa N, Bianco PR, Baskin RJ, Kowalczykowski SC (2005) Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition. Mol Cell 17(5):745–750. https://doi.org/10.1016/j.molcel.2005.02.011
Biosciences G (2002) Gel filtration. Principles and methods. GE Healthcare Bio-Sciences AB, Uppsala
Bianco PR, Stanenas AJ, Liu J, Cohan CS (2012) Fluorescent single-stranded DNA-binding proteins enable in vitro and in vivo studies. Methods Mol Biol 922:235–244. https://doi.org/10.1007/978-1-62703-032-8_18.
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(6):1005–1020. https://doi.org/10.1002/pro.633
Zhao T, Liu Y, Wang Z, He R, Xiang Zhang J, Xu F, Lei M, Deci MB, Nguyen J, Bianco PR (2019) Super-resolution imaging reveals changes in Escherichia coli SSB localization in response to DNA damage. Genes Cells 24(12):814–826. https://doi.org/10.1111/gtc.12729
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Yu, C., Bianco, P.R. (2021). In Vivo Binding of Single-Stranded DNA-Binding Protein to Stalled Replication Fork Helicases. In: Oliveira, M.T. (eds) Single Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 2281. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1290-3_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1290-3_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1289-7
Online ISBN: 978-1-0716-1290-3
eBook Packages: Springer Protocols