Figure 5
From: The single-stranded DNA-binding protein of Deinococcus radiodurans
DNA strand exchange reactions promoted by D. radiodurans RecA and E. coli RecA with SSB titrations. Reactions were carried out as described in the Methods section. Circular single-stranded DNA (css) was preincubated with either D. radiodurans or E. coli RecA. ATP and SSB protein (either D. radiodurans or E. coli SSB protein as indicated) were then added and incubated, followed by the addition of homologous linear double-stranded DNA (lds) which initiated the DNA three-strand exchange reaction. The nicked circular double-stranded DNA product (nc) is distinguishable by agarose gel electrophoresis and quantifiable. Panels A and B show the agarose gel electrophoresis results of reactions promoted by D. radiodurans RecA with various monomer concentrations of D. radiodurans SSB protein and E. coli SSB protein, respectively. These results are quantitated in panel C, with the data from reactions with D. radiodurans SSB (closed circles) and E. coli SSB (closed triangles) coming from Panels A and B respectively. Panel D shows the quantitated results of similar reactions promoted by E. coli RecA with D. radiodurans SSB (closed circle) and E. coli SSB (closed triangle) (agarose gel not shown). Since an SSBDr monomer has two OB folds and an SSBEc monomer has only one, the improved reactions seen in the reactions containing the former protein could simply reflect the higher effective concentration of OB folds when monomeric SSB concentrations are compared. In panels C and D, the dashed lines represent a plot in which the percentage reaction product generated in the reactions using SSBDr are plotted against the actual concentration of OB folds in these reactions (twice the actual concentration of D. radiodurans SSB monomers). The dashed lines (--X--) thus allow a direct comparison of the reactions observed with the D. radiodurans SSB protein with the reactions observed with the E. coli SSB protein (closed triangles). The production of nicked circular double-stranded DNA product is calculated as a percentage of total duplex DNA (the sum of linear double-stranded DNA substrate, nicked circular double-stranded DNA product and any network products near the well).