title: Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.
abstract: Single-stranded DNA-binding protein (SSB) is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated a reagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria. We have demonstrated the utility of the probe in a DNA unwinding reaction using a helicase from Bacillus and for the first time, characterized the B. subtilis SSB's DNA binding mode switching and stoichiometry. The importance of SSB in DNA metabolism is not limited to simply binding and protecting ssDNA during DNA replication, as previously thought. It interacts with an array of partner proteins to coordinate many different aspects of DNA metabolism. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors in different bacteria is critical. Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB.
authors: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors: Matthew
authors: M
authors: Green
authors: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Neville S
authors: NS
authors: Gilhooly
authors: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors: Shahriar
authors: S
authors: Abedeen
authors: School of Biosciences, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, UK.
authors: David J
authors: DJ
authors: Scott
authors: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Mark S
authors: MS
authors: Dillingham
authors: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Electronic address: panos.soultanas@nottingham.ac.uk.
authors: Panos
authors: P
authors: Soultanas
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D008958:Models, Molecular; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D004265:DNA Helicases
keywords: Bacillus subtilis; DNA unwinding; Firmicutes; Fluorescence; Helicases; SSB