sequence homology with EcoSSB (4,5). Since the mitochon- drial SSB proteins contain no part homologous to the C-terminal region of the bacterial SSB proteins (25), this ﬁnd- ing was not unexpected. A chimeric protein composed of the DNA-binding domain of HsmtSSB and the C-terminal third of EcoSSB is not able to take over EcoSSB function in vivo either (36). Therefore, the functional homology of the DNA-binding domains of the tetrameric EcoSSB and the dimeric DraSSB must be larger than the functional homology between the homotetrameric proteins EcoSSB and HsmtSSB. We showed that the binding of DraSSB to ssDNA results in a quench of tryptophan ﬂuorescence by >75% under all salt conditions used. Therefore, we used ﬂuorescence titrations for a detailed characterization of the DNA-binding properties of DraSSB. Independent of salt concentration, the binding afﬁn- ity of DraSSB and poly(dT) is larger than 107 M 1. The binding site size of DraSSB is slightly salt dependent and varies between 54 nt per DraSSB dimer at high and 47 nt at low salt concentrations (0.3 M NaCl and 20 mM KPi, 1 mM NaCl and 1 mM KPi, respectively). For EcoSSB, at least two distinctly different DNA-binding modes have been described (38). Whereas under high salt conditions, 65 nt bind per EcoSSB tetramer with almost 90% ﬂuorescence quench, under low salt conditions 35 nt are sufﬁcient to saturate the protein and quench its ﬂuorescence by only 53%. Therefore, comparison of the DNA-binding properties of DraSSB and EcoSSB gives a slightly reduced binding site size for DraSSB under high salt conditions. The distinctly different binding mode of EcoSSB under low salt conditions could not be observed for DraSSB. Based on the structure of a complex of EcoSSB and two molecules of (dC)35, a model of the low salt binding mode of EcoSSB has been postulated (39). In this model, the Trp54 residues of only two subunits of EcoSSB are involved in DNA binding. The most important difference between the structures of DraSSB and EcoSSB lies in the fact that DraSSB contains two instead of one DNA-binding domain per monomer. These two binding domains (OB-folds) could evolve separately and amino acids known to be involved in DNA binding were conserved more in the C-terminal OB-fold than in the N-terminal one (7). Therefore, it has been speculated that an asymmetric DNA-binding mode, com- parable with the EcoSSB low salt mode, is conserved in DraSSB in which only the C-terminal domains are used for DNA binding (7). Our present data do not support such a speculation since they show that under none of the conditions used a distinct binding mode corresponding to the low salt mode of EcoSSB could be found. The slight reduction of binding site size at high salt compared with EcoSSB is most probably owing to the somewhat smaller overall size of DraSSB. The absence of a low salt binding mode and the fact that DraSSB is functional in E.coli in vivo leads us to speculate that the low salt binding mode does not play a vital role in the life of E.coli. Stopped-ﬂow analysis of binding of DraSSB to poly(dT) showed that the association mechanism and rate constant are quite similar to those of the class of the homotetrameric SSB proteins (10,25). By binding speciﬁcally to ssDNA SSB proteins destabilize DNA double strands. Comparison of DNA melting of poly(dA–dT) in the presence of DraSSB and EcoSSB respect- ively, revealed a weaker destabilization of the double strand by Nucleic Acids Research, 2005, Vol. 33, No. 5 1669 DraSSB than by EcoSSB. Since in this type of experiment the temperature is varied, differences in ssDNA-binding afﬁnity may also reﬂect different temperature dependence of protein stability or DNA-binding afﬁnity. Both GuaHCl denaturation experiments in sedimentation equilibrium and irreversible denaturation of DraSSB at temperatures above 50 C in our DNA melting experiments led to the conclusion that DraSSB is less stable than EcoSSB. Therefore, we decided to measure the binding afﬁnity at room temperature using poly(rU) as a relatively weak binding substrate. In these experiments, no difference in nucleic acid binding afﬁnity of DraSSB and EcoSSB could be detected. Sedimentation velocity experiments under low salt condi- tions showed that DraSSB bound to excess poly(dT) shows a highly asymmetric distribution caused by a large cooperativ- ity of binding. These cooperative complexes are not at equilibrium and redistribute after 96 h of incubation. Such a formation of transiently highly cooperative complexes has also been described for EcoSSB (32). However, the redistribu- tion of the non-randomly bound SSB proteins is much faster in the case of DraSSB. 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