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.

Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction

“…3). In accordance with previous characterisations [15] and under the experimental conditions used, dT70 engages all four monomers forming a fully wrapped complex while dT35 only partially wraps, engaging with 2-3 monomers (Supplementary Fig. 3).…”

“…Wild type SSB was purified using the method previously described [15,16]. The SSB Δ107-171 (CTD removed) construct included an N-terminal histidine tag.…”

“…Data were processed with Primus [15] and ScÅtter [16]. CRYSOL and CRYSON [17] were run in command-line mode to generate theoretical scattering curves using the following adjusted parameters, as defined in the programs manual: /lm 50, /fb 18, /ns 2000.…”

Interactions of the Bacillus subtilis DnaE polymerase with replisomal proteins modulate its activity and fidelity

“…14 nt) prior to the addition of polymerases in the presence and absence of DnaN. Under these conditions, there is more than enough SSB to coat the entire ssM13mp18 DNA template [ 50 ]. In the presence of SSB, the activity of DnaE was stimulated substantially, producing large amounts of nascent DNA greater than 1500 nt in 1 min ( figure 4 c (i)).…”

Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA

“…Fluorescently labeled versions of Ec -SSB and Bacillus subtilis SSB have been developed as real-time probes for ssDNA in biochemical assays[ 22 , 27 , 28 ]. However, one has to take into account the buffer-dependent variations and cooperative behavior of these SSBs to adequately interpret results.…”