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.
Highlights
The benefit of fluorescent reagentless biosensors to track enzymatic reactions, with minimal disruption to activity has been extensively discussed (Galban et al, 2012)
One class of biosensors use protein–fluorophore adducts to create highly specific and sensitive probes that exploit the binding characteristics of the protein component (Gilardi et al, 1994; Salins et al, 2001; Kunzelmann and Webb, 2011) Helicase unwinding is stimulated by proteins that bind to the unwound (deoxynucleic acid) (DNA) strands and prevent reannealing (Dillingham et al, 1999)
Single-stranded DNA-binding protein (SSB) binds (single stranded DNA) (ssDNA) in a groove that extends around the whole N-terminal domain creating many potentially appropriate fluorophore attachment sites
Summary
The benefit of fluorescent reagentless biosensors to track enzymatic reactions, with minimal disruption to activity has been extensively discussed (Galban et al, 2012). In E. coli and in B. subtilis, SSB has been shown to physically interact with at least 12 proteins facilitating the functional organization of replication forks (Costes et al, 2010), and so the use of cognate SSB proteins as reagentless biosensors is desirable. This essential function of SSB is well conserved throughout the three domains of life (Glassberg et al, 1979). The RNA primer is passed from DnaG to DnaE via a direct physical interaction between the two proteins (Rannou et al, 2013) Such critical functional differences mean it is important to develop a cognate SSB probe that is compatible with the biological system under study. We present a cheap method with commercially available fluorophores to produce a B. subtilis SSB biosensor, have characterized its properties and confirmed its use in a grampositive specific helicase reaction
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