ssDNA binding properties The purified SSB proteins were analyzed for single-stranded DNA binding activity. In these experiments, a fixed concentration of (dT)n (n = 35, 76 or 120 nucleotides in length) were incubated with various SSB concentrations and the resulting complexes were analyzed by agarose gel electrophoresis (Figure 3). When dT35 was incubated with increasing concentrations of each of the SSB proteins, a single band of reduced mobility was LY333531 observed and remained constant even at a higher protein RXDX-101 concentration (complex I). A band with the same mobility was observed for (dT)76 at a low protein concentration, but a second band with a lower mobility was observed at a high protein concentration
(complex II). When SSB:dT120 click here complexes were analyzed, a third band with a lower mobility was detected (complex
III). This implies that the length of ssDNA required for efficient protein binding is less than 35 nucleotides long. Figure 3 Binding of SSB proteins to oligo (dT). Fixed quantities (10 pmol) of 5′-end fluorescein-labelled oligonucleotides (dT)35, (dT)76 and (dT)120 were incubated with 50, 100 and 200 pmol of the SSB proteins in 20 μl reaction mixtures for 10 min at 25°C. Symbols I, II and III describe SSB:dT complexes. In order to explore the binding properties of all the proteins in question further, we used fluorescence spectroscopy. All the bacterial SSBs which have been studied to date have shown a dramatic decrease of tryptophan fluorescence when binding to ssDNA. With an excitation wavelength of 295 nm, the emission spectrum of SSB proteins at 25°C reached its maximum at 348 nm, which is consistent with tryptophan fluorescence. On the addition of a saturating quantity of (dT)76, the intrinsic fluorescence at 348 nm was quenched by 93±3% for the DpsSSB, FpsSSB, ParSSB, PcrSSB, and PtoSSB, by 90±3% for the PprSSB, and by 81±3% for the PinSSB. It was salt independent. The estimated binding site was determined as being approximately 30 ± 2 nucleotides long for the PinSSB, 31 ± 2 nucleotides
for the DpsSSB and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB, and PtoSSB. Practically no binding mode transition was observed when changing the Sirolimus solubility dmso ionic strength from low to high salt (Figure 4). However, for the FpsSSB, a binding-mode transition of 31 ± 2 nucleotides at low salt concentrations and 45 ± 2 at high ones was observed. Figure 4 Inverse fluorescence titration of SSB proteins with oligo(dT) 76 . The 1.5 nmol samples of the SSB proteins under study were titrated with (dT)76 at 2 mM (Δ), 100 mM (□) and 300 mM (○) NaCl binding buffer. dsDNA melting point destabilization A destabilization of DNA double strands in the presence of SSB must be expected as a thermodynamic consequence of SSB proteins binding specifically to ssDNA and not to dsDNA.