Table 1.
Signal changes of adducts of PfSSB variants and fluorophore dyes.
Fig 1.
Fluorescent spectra of DCC-PfSSB.
Excitation and emission spectra of 250 nM DCC-PfSSB with and without 595 nM dT70 in high salt buffer. The excitation spectra had emission at 475 nm; emission spectra had excitation at 432 nm. The spectra were normalized to one at the emission maximum in the absence of DNA.
Fig 2.
Titrations of different ssDNA lengths with DCC-PfSSB.
(A) Titrations of dT70 (triangles), dT35 (circles) and poly(dT) (diamonds) into 250 nM DCC-PfSSB. Upper panel were in high salt, lower panel in low salt buffer. Data were fit for tight binding, a linear increase followed by a constant fluorescence with floating breakpoint, or in the case of dT35, two linear increases. (B) dT70 titrations into 250 nM DCC-PfSSB in high salt buffer with and without equimolar unlabeled PfSSB. Data were fit to Eq 1, as described in the Materials and Methods. (C) Titrations with dT70 at low DCC-PfSSB in high salt buffer. At 2.5 nM (main panel) the data were fit to a linear increase followed by a constant fluorescence. At 125 pM (inset), the data from a single titration were fit to a quadratic binding equation (Eq 2), giving a Kd of 1.2 ± 0.5 pM. No correction to amplitudes was made for background scatter, which is the likely cause of the apparently lower fluorescence change.
Fig 3.
DCC-PfSSB association kinetics with excess ssDNA.
(A) Fluorescence time courses of 5 nM DCC-PfSSB following mixing with dT70 in large excess and in high salt buffer. dT70 was at the nanomolar concentrations shown. (B) The data were fit to double exponentials: the fast rate constant increased linearly with concentration, giving an association rate constant of 324 ± 14 μM-1s-1 for dT70. Measurements with dT35 gave 127± 3 μM-1s-1 and 137 ± 6 μM-1s-1 for poly(dT). The intercept was too close to zero to give an accurate measure of the dissociation rate constant. (C) Association kinetics of dT70, dT35 and poly(dT) as in B but in low salt conditions. Time courses were fit to double exponentials. The association rate constants, determined from the linear fits of the fast phase, were 199 ± 9 μM-1s-1 for dT70, 34 ± 1 μM-1s-1 for dT35, and 53 ± 2 μM 1s-1 for poly(dT).
Fig 4.
Association kinetics of ssDNA with excess DCC-PfSSB.
The measurements were in high salt buffer with the ssDNA at 10% of the SSB concentration. (A) Example time courses for with DCC-PfSSB at the micromolar concentrations shown with dT70, dT35 and poly(dT) at 10-fold less concentration. To show on single panels traces are normalized to a total fluorescence change of 1. (B) Linear dependence of the observed rate constant of the fast phase on the concentration of the DCC-PfSSB, giving second order rate constants for dT70, 397 ± 31 μM-1s-1, poly(dT) 146 ± 13 μM-1s-1 and dT35 290 ± 28 μM-1s-1.
Fig 5.
Dissociation kinetics for DCC-PfSSB.dT70.
(A) The measurement were done in high salt buffer by premixing DCC-PfSSB, with a slight excess of dT70 to form the DCC-PfSSB·ssDNA complex before mixing with varying concentrations of wtSSB and following the fluorescence time course. The final concentrations in the reaction mix were 10 nM DCC-PfSSB, 12.5 nM dT70 and wtSSB at the nanomolar concentrations shown. (B) Dependence of dissociation kinetics on the wtSSB concentration. Time courses were fit to single exponentials and a linear fit of observed rate constants (kobs) gave the y intercept, -0.0001 ± 0.0009 s-1 and the gradient as 0.208 ± 0.003 μM-1s-1. The observed rate constants remain linearly dependent on [wtSSB] at higher concentrations (inset). (C) Model for mechanism of dissociation with either an associative pathway whereby wtSSB binds to the DNA before DCC-PfSSB dissociates (upper) or simple dissociation (lower). Steps are numbered so the forward and reverse rate constants for step i are k+i and k-I, respectively.
Fig 6.
AddAB helicase assay with linear dsDNA using DCC-PfSSB as a biosensor.
(A) Example fluorescence traces of AddAB different dsDNA lengths (shown in base pairs) using DCC-PfSSB as a reporter for production of ssDNA. The inset shows a cartoon of the complex before unwinding: S is streptavidin; B, biotin; H, helicase. (B) Linear fit of unwinding durations, plotted against the dsDNA lengths. The fit for the durations has gradient of 0.92 ± 0.02 s kb-1, which gives an unwinding rate of 1082 bp s-1. The inset shows the fluorescence changes plotted against dsDNA lengths.