and Thermus thermophilus (6,8,9). Comparing the DraSSB sequence with other bacterial SSB sequences [e.g. from E.coli (26)] shows that the N-terminal DNA-binding domain (the OB-fold) occurs twice in tandem in DraSSB and the other Thermus SSBs while it occurs only once in the EcoSSB gene. A possible explanation for this difference is a gene duplication event that occurred during the evolution of the Thermus group SSBs. The highly conserved acidic C-terminus with the consensus sequence DDDIPF is also present in DraSSB (EDDLPF). Structural characterization of DraSSB DraSSB protein overexpressed in E.coli was puriﬁed to homo- geneity (>97%). Figure 1 shows a sedimentation velocity experiment in the analytical ultracentrifuge. Analysis of the sedimenting boundary using Lamm’s differential equation yields a sedimentation rate constant of s20 C,W = 3.95S and a diffusion constant of D20 C,W = 6.3 · 10 11 m2 s 1. Com- bining these two values with the partial speciﬁc volume of 7.23 · 10 4 m3 kg 1 calculated from the amino acid com- position (21) gives a molar mass of 55 kg/mol. The molar mass of the covalent protein chain of DraSSB is calculated to be 32.7 kg/mol. Since the diffusion coefﬁcient evaluated from the broadening of the sedimenting boundary tends to be overes- timated owing to artiﬁcial broadening effects (27), a molar mass derived from sedimentation and diffusion coefﬁcients constitutes a lower limit and the ﬁnding, thus, is a clear indica- tion that the native state of DraSSB is a homodimer. Using the molar mass of the dimer as calculated from the amino acid composition, the sedimentation rate constant of 3.95S leads to a frictional ratio of 1.5. Since for globular hydrated proteins this frictional ratio is expected to be 1.1–1.2 (27), a value of 1.5 shows the protein to be either strongly asymmetric or globular with several protuberances. For EcoSSB, it has been shown that the large frictional coefﬁcient of 1.42 is reduced to 1.25 when cleaving off the C-terminal third of the protein (11). Thus, it seems likely that similar to EcoSSB the C-terminal part of DraSSB extends into the solution. This is also supported by gel ﬁltration experiments, which suggest Nucleic Acids Research, 2005, Vol. 33, No. 5 1665 DraSSB to be asymmetric (9), and by recent X-ray crystallo- graphic studies where the C-terminal part of DraSSB could not be localized (7), probably owing to large disorder. Sedimentation equilibrium in the analytical ultracentrifuge gave a molar mass of M = 62 – 2 kg/mol with no indication of multiple species or aggregation, conﬁrming the result obtained for a differently prepared DraSSB before (9) and clearly show- ing again that DraSSB forms homodimers. We have tested the conformational stability of DraSSB by sedimentation equilibrium experiments in the presence of dif- ferent concentrations of GuaHCl. GuaHCl denaturation, mon- itored by changes in the apparent molar mass (Figure 2), results in a transition midpoint at 1 M denaturant. DraSSB, therefore, is clearly more sensitive to GuaHCl-induced dena- turation than EcoSSB whose transition midpoint is at 1.7 M (Figure 2). Thus, the folded state of DraSSB is less stable than the bacterial prototype EcoSSB. This relative instability is also reﬂected in the temperature stability of these two proteins. While DNA melting experiments with EcoSSB can be performed up to 65 C, DraSSB irreversibly denatures at temperatures above 50 C. In vivo detection of SSB in D.radiodurans It remains to be demonstrated that the ORF cloned and expressed in E.coli encodes a genuine protein of D.radiodurans. Western blot analysis shows that a polyclonal anti-EcoSSB serum cross-reacts with cloned DraSSB and detects a correctly sized protein in a total protein extract from D.radiodurans (Figure 3A). DraSSB can replace EcoSSB in vivo Though different in oligomerization, EcoSSB and DraSSB share the ssDNA-binding folds (OB-fold) (7) and the C-terminal region, for which important protein–protein inter- actions have been shown (28–30). Therefore, we tested whether DraSSB can take over the EcoSSB function in vivo using an ssb deletion strain (RDP268). In RDP268, the chromosomal ssb gene has been replaced by a kanamycin resistance and the vital SSB function is supplied by EcoSSB encoded on the chloramphenicol resistance conferring plasmid pACYCssb (15). After transformation of these cells using pSF1-DraSSB, Figure 1. Sedimentation of DraSSB in the analytical ultracentrifuge. A sample containing 7.5 mM DraSSB in 0.3 M NaCl and 20 mM KPi was centrifuged at 20 C and 60 000 r.p.m.; scans were taken every 8 min. Smooth lines correspond to theoretical traces for a sedimentation coefficient of s20 C,W = 3.95S and a molar mass of 55 kg/mol (D20 C,W = 6.3 · 10 11 m2 s 1) calculated with a numerically integrated Lamm differential equation (cf. Materials and Methods). Figure 2. Guanidinium-induced unfolding of 4 mM DraSSB (circles) and 3.5 mM EcoSSB (triangles) in 0.3 M NaCl and 20 mM KPi. Apparent molar masses (cf. Materials and Methods) were determined from sedimentation equilibrium centrifugation at 20 C and 14 000 r.p.m. Note the different axes.