Nucleic Acids Research, 2005, Vol. 33, No. 5 1667 Figure 5. Stopped-flow kinetics of the association of DraSSB with poly(dT) at 0.3 M NaCl, 20 mM KPi, 100 p.p.m. Tween-20. Concentrations of DraSSB and poly(dT) were (a) 0.8 and 40 mM (b) 0.4 and 20 mM (c) 0.2 and 10 mM (d) 0.1 and 5 mM (e) 50 nM and 2.5 mM (f) 25 nM and 1.25 mM (g) 12.5 nM and 0.625 mM, respectively. The curves were normalized to show equal amplitudes. Smooth lines represent the theoretical time courses for the mechanism described in the text (10) using an association rate constant kass of 2.1 · 108 M 1 s 1, a binding site size n of 55 and an initial binding site length of 11 nt. leads to a non-random distribution of ssDNA/SSB complexes. Gel electrophoresis of 50% saturated ssM13mp7 DNA showed two different species that were attributed to fully saturated and non-saturated ssDNA (32). Such a bifurcation of SSB distribution on long ssDNA can also be seen in analytical ultracentrifugation experiments with poly(dT) and SSB. In Figure 6, the c(s) distribution (19) of a poly(dT)/DraSSB mixture at low salt conditions (5 mM NaCl, 5 mM KPi and 0.87 M glycerol) is shown. Sedimenting the mixture shortly (1 h) after preparation leads to two different complexes with 7S and 13S, respectively. Glycerol had to be added to avoid protein aggregation and owing to its viscosity increment free poly(dT) and DraSSB sediment with 4S and 3S, respectively, in this buffer. The non-random distribution of DraSSB molecules on the poly(dT) strands is a consequence of the highly cooperative binding. If, however, the mixture of DraSSB and poly(dT) is incubated for longer periods (24–96 h), the two species merge and after 4 days incubation at room temperature only a single species sedimenting with an intermediate sedimentation constant (10S) can be detected (Figure 6). Therefore, the DraSSB molecules that initially bind in a non-random distribution redistribute very slowly on the poly(dT) strands. This slow rearrangement reﬂects a very large lifetime of DraSSB bound cooperatively to poly(dT). For EcoSSB under the same buffer conditions, we could also observe non-random distribution on poly(dT) by analytical ultracentrifugation conﬁrming gel electrophor- etic experiments reported previously (32). However, redistribution of EcoSSB is much slower and even after pro- longed incubation (2 weeks), we could not reach fully random distribution (data not shown). Thus, cooperatively bound DraSSB more easily redistributes on poly(dT) than EcoSSB. At high salt conditions (0.3 M NaCl and 20 mM KPi), both SSB proteins showed a random distribution on the poly(dT) strands independent of incubation time (data not shown). Therefore high DNA-binding cooperativity under high salt conditions could be excluded. Figure 6. DraSSB shows transient cooperativity by binding to ssDNA at low salt. Mixtures of 0.35 mM DraSSB and 35 mM poly(dT) in 5 mM NaCl, 5 mM KPi and 0.87 M glycerol were analyzed in sedimentation velocity experiments in the analytical ultracentrifuge (23 000 r.p.m., 20 C, An50Ti rotor) after 1 h (solid line) and 96 h (dotted line) incubation at room temperature. The differential sedimentation coefficient distributions were obtained using the program SEDFIT (19). The initially non-randomly distributed DraSSB mole- cules redistribute within 96 h on the poly(dT) strands. Figure 7. DraSSB and EcoSSB show different influence on the melting behavior of poly(dA–dT). Samples containing 38 mM nucleotides of poly(dA–dT) in a buffer containing 75 mM NaCl and 20 mM KPi were melted in the presence of 3.25 mM EcoSSB (a), 3.25 mM DraSSB (b) and in the absence of protein (c). As a thermodynamic consequence of SSB proteins binding speciﬁcally to ssDNA and not to dsDNA, a destabilization of DNA double strands in the presence of SSB must be expected. Figure 7 shows that the melting temperature of poly(dA–dT) is decreased from 59 to 42 C by excess amounts of DraSSB. Under the same conditions, EcoSSB decreases the melting temperature from 59 to 37 C, indicating a weaker afﬁnity of DraSSB for ssDNA under the conditions of the melting experiment. Protein–protein interactions of DraSSB As described earlier, the C-terminal region of EcoSSB is responsible for protein–protein interactions (28–30). Since the corresponding sequences of DraSSB and EcoSSB are highly homologous, there is a high possibility of correspond- ing interactions in D.radiodurans (9). One of the proteins functionally interacting with the C-terminus of EcoSSB is