Abstract
The Bacillus subtilis phage Phi29 has a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA). Phi29 single-stranded DNA-binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated during the Phi29 genome replication, stimulating the DNA elongation rate. Here, we describe some protocols to evaluate the effect of Phi29 SSB mutants on the DNA elongation rate and their unwinding activity during replication by Phi29 DNA polymerase using as substrate TP-DNA and also singly primed M13 DNA.
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References
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Acknowledgments
This chapter is dedicated to the memory of Professor Margarita Salas. This work was supported by grant BFU2014-52656-P (to Margarita Salas) from the Spanish Ministry of Economy and Competitiveness and institutional grants from the Fundación Ramón Areces and Banco Santander to the Centro de Biología Molecular “Severo Ochoa.”
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del Prado, A., Salas, M. (2021). Strand Displacement and Unwinding Assays to Study the Concerted Action of the DNA Polymerase and SSB During Phi29 TP-DNA Replication. 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_22
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DOI: https://doi.org/10.1007/978-1-0716-1290-3_22
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