The single-stranded DNA-binding protein gp2.5 of bacteriophage T7 plays myriad functions in the replication of phage genomes. In addition to interacting with ssDNA, gp2.5 binds to the T7 DNA polymerase and primase/helicase proteins, regulating their enzymatic activities. Here we describe in vitro methods to examine the effects of gp2.5 on primer synthesis and extension by the T7 replisome.
Hamdan SM, Richardson CC (2009) Motors, switches, and contacts in the replisome. Annu Rev Biochem 78:205–243. https://doi.org/10.1146/annurev.biochem.78.072407.103248
[DOI:
10.1146/annurev.biochem.78.072407.103248]
Lee SJ, Richardson CC (2011) Choreography of bacteriophage T7 DNA replication. Curr Opin Chem Biol 15:580–586. https://doi.org/10.1016/j.cbpa.2011.07.024
[DOI:
10.1016/j.cbpa.2011.07.024]
Kulczyk AW, Richardson CC (2016) The Replication System of Bacteriophage T7. Enzyme 39:89–136. https://doi.org/10.1016/bs.enz.2016.02.001
[DOI:
10.1016/bs.enz.2016.02.001]
Lee J, Chastain PD 2nd, Griffith JD, Richardson CC (2002) Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins. J Mol Biol 316:19–34. https://doi.org/10.1006/jmbi.2001.5325
[DOI:
10.1006/jmbi.2001.5325]
Lee J, Chastain PD 2nd, Kusakabe T, Griffith JD, Richardson CC (1998) Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol Cell 1:1001–1010
[DOI:
10.1016/S1097-2765(00)80100-8]
Hernandez AJ, Richardson CC (2019) Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein. Semin Cell Dev Biol 86:92–101. https://doi.org/10.1016/j.semcdb.2018.03.018
[DOI:
10.1016/j.semcdb.2018.03.018]
Nakai H, Richardson CC (1988) The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7. J Biol Chem 263:9831–9839
[DOI:
10.1016/S0021-9258(19)81592-2]
Hernandez AJ, Lee SJ, Richardson CC (2016) Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome. Proc Natl Acad Sci U S A 113:5916–5921. https://doi.org/10.1073/pnas.1604894113
[DOI:
10.1073/pnas.1604894113]
Kim YT, Tabor S, Bortner C, Griffith JD, Richardson CC (1992) Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein. J Biol Chem 267:15022–15031
[DOI:
10.1016/S0021-9258(18)42141-2]
Tabor S, Huber HE, Richardson CC (1987) Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem 262:16212–16223
[DOI:
10.1016/S0021-9258(18)47718-6]
Tabor S, Richardson CC (1981) Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc Natl Acad Sci U S A 78:205–209
[DOI:
10.1073/pnas.78.1.205]
Akabayov B, Akabayov SR, Lee SJ, Tabor S, Kulczyk AW, Richardson CC (2010) Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin. Proc Natl Acad Sci U S A 107:15033–15038. https://doi.org/10.1073/pnas.1010141107
[DOI:
10.1073/pnas.1010141107]
Cao W, De La Cruz EM (2013) Quantitative full time course analysis of nonlinear enzyme cycling kinetics. Sci Rep 3:2658. https://doi.org/10.1038/srep02658
[DOI:
10.1038/srep02658]
Johnson KA (2013) A century of enzyme kinetic analysis, 1913 to 2013. FEBS Lett 587:2753–2766. https://doi.org/10.1016/j.febslet.2013.07.012
[DOI:
10.1016/j.febslet.2013.07.012]
Johnson KA (2009) Fitting enzyme kinetic data with KinTek global kinetic explorer. Methods Enzymol 467:601–626. https://doi.org/10.1016/S0076-6879(09)67023-3
[DOI:
10.1016/S0076-6879(09)67023-3]
DNA Primers
DNA, Single-Stranded
DNA, Viral
DNA-Binding Proteins
Viral Proteins
gene 2.5 protein, Enterobacteria phage T7
DNA-Directed DNA Polymerase
