1668 Nucleic Acids Research, 2005, Vol. 33, No. 5 Figure 8. Interaction of DraSSB and E.coli DNA polymerase III c subunit detected by analytical ultracentrifugation. Analytical sedimentation velocity centrifugation of 2.65 mM DraSSB (dotted line) and a mixture of 2.65 mM DraSSB and 31.8 mM c (solid line) in 0.3 M NaCl, 20 mM KPi at 20 C and 50 000 r.p.m. Differential sedimentation coefficient distributions were obtained using the program SEDFIT (19). In the presence of c, a complex is formed which sediments faster than any of the free proteins. Inset: for different mixtures concentrations of free and bound c were determined from the areas under the separated peaks of the differential sedimentation coefficient distributions and were used to construct a binding isotherm. The solid line represents the best non-linear least squares fit for independent binding of 2.2 c molecules to a DraSSB dimer with K = 7.4 · 104 M 1. the c subunit of E.coli DNA polymerase III, for which qual- itative and quantitative studies of the interaction have been reported previously (29,30). Thus, and since we have shown that DraSSB can take over EcoSSB function in E.coli, we decided to test protein–protein interactions of DraSSB using c protein of E.coli as a ligand. Figure 8 shows a c(s) distribution (19) of the sedimentation of DraSSB in the pres- ence and absence of c. Binding of c leads to an increase of the sedimentation coefﬁcient from 3.6S for free DraSSB to 4S. A binding isotherm constructed from an analysis of boundary heights shows binding of 2 c proteins to a DraSSB dimer with an approximate afﬁnity of 105 M 1 (Figure 8, inset). There- fore, in DraSSB all C-terminal regions are accessible for c binding. DISCUSSION Sequence similarity classiﬁes the ssDNA-binding protein of D.radiodurans to belong to the class of homotetrameric SSB proteins. However, the protomer of DraSSB is nearly twice the size of typical homotetrameric SSB protein subunits. Whereas the protomers of the tetrameric SSB proteins contain only one DNA-binding domain (OB-fold), the SSB protein from D.radiodurans and from other species of the Thermus group contain two OB-folds (7), indicating a gene duplication in the evolution of the Thermus group of bacteria. From D.radiodurans cells (strain DSM20539), we ampliﬁed the ssb gene by PCR. Analysis of the DNA sequence resulted in an ORF coding for 301 amino acids and revealed sequen- cing errors in the genomic sequence originally published (34). These errors were recently reported by others also (9). After cloning of the DraSSB gene, we expressed the protein in E.coli and puriﬁed it to homogeneity. By western blot analysis, we could show that recombinant DraSSB protein cross-reacts with anti-EcoSSB antibodies and we identiﬁed the same protein in a total protein extract of D.radiodurans. Sedimentation equilibrium experiments of DraSSB gave a molar mass of 62 kg/mol, clearly showing that DraSSB forms dimers in solution. This is in excellent agreement with the results of (9). Sedimentation velocity analysis revealed a fric- tional ratio of 1.5, which indicates that the DraSSB dimer must be either strongly asymmetric or globular with protrusions. DraSSB, like other proteins from the class of homotetrameric SSB proteins, contains in its C-terminal part a proline- and glycine-rich region, which is expected to be very ﬂexible. Therefore, it seems probable that the high frictional ratio of DraSSB, as well as of the other bacterial SSB proteins (11), is caused by the extension of this region into the solution. Sim- ilarly to the other homologs, in DraSSB the proline- and glycine-rich region is succeeded by the last 10 amino acids, which are highly conserved in this class. For EcoSSB, it could be shown that this region is responsible for the interaction with other proteins involved in DNA metabolism (28–30,35). It has been speculated that the glycine- and proline-rich region acts as a spacer between the DNA-binding domain and the nega- tively charged region of the last 10 amino acids (36). Thus, the formation of complexes between SSB proteins and their inter- action partners could be facilitated by an easy access of this region. We showed that a DraSSB dimer interacts with up to two molecules of E.coli DNA polymerase III c subunit. Hav- ing four protomers EcoSSB protein binds up to four c molecules (30). Thus, both C-terminal regions of the dimeric DraSSB are accessible for the interaction with other proteins. The afﬁnity of the DraSSB/c interaction is 105 M 1, similar to the afﬁnity of the EcoSSB/c interaction (30), despite the fact that E.coli DNA polymerase III c subunit is not a natural interaction partner of DraSSB in vivo. This again demonstrates that interactions of the bacterial SSB proteins with other proteins are evolutionarily conserved and that the conserved region of the last 10 amino acids plays a crucial role in these interactions (36). It remains to be noted that in silico analysis of D.radiodurans genome failed to reveal an ORF coding for a protein homologous to the c subunit of DNA polymerase III from E.coli. Other proteins known to interact with bacterial SSB proteins [uracil DNA glycosylase (35) and primase (37)], however, have been identiﬁed in silico (Q9RWH9, Q9RWR5, respectively). Thus, it seems likely that evolutionary diver- gence led to a c protein in D.radiodurans that could not be detected by the present in silico methods. EcoSSB is essential for the survival of the E.coli cell (15), and the conserved region of the last 10 amino acids is neces- sary for EcoSSB functioning in vivo (36). Using an ssb dele- tion strain, we could show that DraSSB can take over the function of EcoSSB in vivo. Since DraSSB as a dimer contains only two instead of four C-termini, two C-termini seem to sufﬁce for the in vivo function of SSB. It has been speculated that the loss of two of the four C-termini in DraSSB may lead to functional differences (9). Our data clearly show that such functional differences, even if they exist, do not play an important role in the function of SSB in vivo. The tetrameric human mitochondrial SSB (HsmtSSB) protein cannot complement an EcoSSB deletion mutant strain (36), although it is structurally strongly related and shares 32%