1666 Nucleic Acids Research, 2005, Vol. 33, No. 5 which encodes resistance against ampicillin, and subsequent inoculations (cf. Materials and Methods), we could isolate clones that showed resistance against ampicillin and kanamy- cin but not against chloramphenicol. These clones must have lost the pACYCssb plasmid encoding for EcoSSB. SSB expression was examined by western blot analysis using an anti-EcoSSB serum as a primary antibody (Figure 3B).While in RDP268 EcoSSB could be detected, the chloramphenicol- sensitive clones showed an expression of only DraSSB (32.7 kDa) but not of EcoSSB (18.8 kDa). DraSSB binding to ssDNA Whenever two macromolecules interact, the resulting complex will have a larger mass than any of the single components and, thus, it will sediment faster than the single species. Binding SSB proteins to polymeric ssDNA [poly(dT)] results in a dramatic increase in sedimentation rate. At 0.3 M NaCl, 20 mM KPi poly(dT) of 1400 nt in length has a sedimenta- tion coefﬁcient of 8S. Addition of different amounts of Figure 3. Verification of cloned DraSSB as a genuine D.radiodurans protein (A) and complementation assay (B). Samples were boiled in 1% SDS sample buffer, proteins were separated in Tricin SDS–PAGE (40), blotted on PVDF membrane and immunologically detected with a polyclonal rabbit anti-EcoSSB serum. (A) Cell extract from D.radiodurans (lane 1) compared with 200 ng purified recombinant DraSSB (lane 2). (B) Lane 3, RDP268 (pSF1-DraSSB) cells express only DraSSB; lane 4, RDP268 (pACYCssb) cells express only EcoSSB; and lane 5, purified recombinant EcoSSB (15 ng). Lane 3 and lane 4 contain equal amounts of cells. DraSSB (3.6S) to this poly(dT) leads to an increase of this sedimentation coefﬁcient up to 26S at saturation. Saturation point is reached at a stoichiometry of 50 – 5 nt per DraSSB dimer corresponding to a complex of 28 SSB proteins on each poly(dT) strand (data not shown). All homotetrameric SSB proteins studied so far show a dramatic decrease of tryptophan ﬂuorescence when binding to ssDNA (25,31,32). Figure 4 shows titrations of DraSSB with poly(dT) at high salt (0.3 M NaCl and 20 mM KPi) and low salt (1 mM NaCl and 1 mM KPi) conditions. For com- parison, similar titrations of EcoSSB are shown. Under high salt conditions, binding isotherms give a stoichiometry of 54 – 2 nt/DraSSB(dimer) and tryptophan ﬂuorescence is quenched by 87 – 2%. At low salt conditions, the stoichi- ometry is reduced to 47 – 2 nt/DraSSB(dimer) and the ﬂuor- escence quench is diminished to 77 – 2%. In both the cases, the cooperative afﬁnity is estimated to be in the range of 107–108 M 1. Stopped-ﬂow analysis of the association of DraSSB to poly(dT) at high salt (0.3 M NaCl and 20 mM KPi) (Figure 5) is compatible with a binding mechanism where the initial binding of the protein uses a short stretch of nuc- leotides that are bound in a bimolecular mechanism with an association rate constant of 2.1 · 108 M 1 s 1. Subsequently, the protein–nucleic acid complex rearranges in a monomo- lecular reaction to reach its ﬁnal binding site size (cf. Materials and Methods). The association mechanism and rate constant are in accordance with the other homotetrameric SSB proteins investigated so far (10,25). Since binding of SSB to poly(dT) is too strong to reliably determine binding afﬁnities by ﬂuorescence titrations, we used poly(rU) as a weakly binding substrate (33) to compare the binding strength of EcoSSB and DraSSB. Fluorescence titra- tions at high salt (0.2 M NaCl and 20 mM KPi) showed no detectable difference in the binding afﬁnity of these proteins (data not shown). For low salt concentrations (22 mM NaCl), it has been reported that binding of EcoSSB to long ssDNA is highly cooperative. At intermediate saturation, such cooperativity Figure 4. Fluorescence titrations of DraSSB and EcoSSB with poly(dT) at 22 C and different salt conditions. Solid lines are theoretical binding isotherms for the binding of a multidentate ligand to a linear polymer (23) with binding site size n and cooperative affinity w K as indicated below. (A) Circles: 0.375 mM DraSSB in 0.3 M NaCl, 20 mM KPi, 100 p.p.m. Tween-20; n = 53.9, w K = 1.2 · 108 M 1, Qf = 86.2%. Triangles: 0.375 mM DraSSB in 1 mM NaCl, 1 mM KPi, 100 p.p.m. Tween-20; n = 47.5, w K = 5 · 107 M 1, Qf = 74.9%. (B) Circles: 0.29 mM EcoSSB in 0.3 M NaCl, 20 mM KPi, 0.1 mM EDTA, 100 p.p.m. Tween-20; n = 63.7, w K = 1.5 · 108 M 1, Qf = 92%. Triangles: 0.44 mM EcoSSB in 1 mM NaCl, 1 mM KPi, 0.1 mM EDTA, 100 p.p.m. Tween-20; n = 39.5, w K = 9.3 · 106 M 1, Qf = 70%.