1664 Nucleic Acids Research, 2005, Vol. 33, No. 5 phosphatase chemiluminescence reaction (CDP-Star, Tropix). Exposed X-ray ﬁlms were digitized. Analytical ultracentrifugation Analytical ultracentrifugation was performed in a Beckman XL-A analytical ultracentrifuge using an An50Ti or An60Ti rotor at 20 C. Concentration proﬁles were measured with UV absorption scanning optics of the centrifuge. Sedimentation velocity Sedimentation velocity experiments were carried out in double-sector centerpieces at 20 000–60 000 r.p.m. Sedimen- tation rate constants were obtained by analyzing the movement of the sedimenting boundary or by ﬁtting a numerical solution of Lamm’s differential equation (17) to the concentration pro- ﬁles using the BPCFIT software package (18). For analysis of interactions, data were evaluated with SEDFIT (19) to yield diffusion-corrected differential sedimentation coefﬁcient dis- tributions c(s). The areas under separate peaks of such a dis- tribution are a measure of the absorption of the differently sedimenting components and can be analyzed to yield a bind- ing isotherm (20). For hydrodynamic analysis, s-values were corrected to s20 C,W. Since the partial speciﬁc volume of complexes of different macromolecules with unknown stoi- chiometry cannot be calculated, such a correction could not be performed in these cases and uncorrected s-values had to be used. Sedimentation equilibrium Sedimentation equilibrium experiments for molar mass determination were carried out with 120 ml samples in 6-channel-centerpieces in an An50Ti rotor running at 20 C at 14 000 r.p.m. until no change in concentration distribution could be observed for at least 12 h. Scans from these 12 h were averaged and apparent molar masses were evaluated by ﬁtting the concentration gradient with Mð1 nrÞw2ðx2 x20Þ AðxÞ ¼ Aoffset þ Aðx0Þ e 2RT where x is the distance from centre of rotation, w is the angular velocity of the rotor, v is the partial speciﬁc volume of the solute calculated from amino acid composition (21), r is the density of the solution, A(x) is the absorption at position x, A(x0) is the absorption of the macromolecule at x0 and Aoffset is the absorption of the buffer measured near the meniscus after sedimenting the protein for 8 h at 44 000 r.p.m. Fitting was carried out with BPCFIT (18). Protein stability against dena- turation by guanidine hydrochloride (GuaHCl) was measured in sedimentation equilibrium experiments using different con- centrations of GuaHCl. Note that guanidinium denatured pro- teins have a molar mass somewhat higher than the molar mass of the covalent protein chain owing to preferential binding of the denaturant. DNA melting curves DNA melting curves were measured in a DMR10 (Zeiss, Germany) spectrophotometer as described previously (22) using a heating rate of 20 K/h. The experiments were carried out with 38 mM nucleotides of poly(dA–dT) in 75 mM NaCl, 20 mM KPi. As long as the temperature did not exceed 50 C, no signiﬁcant differences between heating and cooling curves could be observed, conﬁrming the reversibility of melting. Above 50 C, irreversible heat denaturation of DraSSB occurred. Fluorescence titrations Fluorescence titrations were carried out in a Schoeffel RRS1000 spectroﬂuorimeter as described previously (23). Excitation wavelength was 295 nm and emission was detected at 350 nm. To avoid inner ﬁlter effects, concentrations of protein and single-stranded nucleic acid were chosen such that the total absorption of the solution did not exceed 0.05 throughout the titration. After each addition, the solution was allowed to equilibrate between 60 and 600 s until no ﬂuores- cence change could be observed any longer. Binding curve analysis was carried out using the model of Schwarz and Watanabe (24) with n as binding site size, w K as cooperative binding afﬁnity and ﬂuorescence quench Qf as parameters. Fluorescence quench is deﬁned as 1 Fbound/Ffree, where Ffree and Fbound denote the ﬂuorescence intensities measured for free and nucleic acid bound protein, respectively. Stopped-flow kinetics Stopped ﬂow kinetics for the determination of the association rate constant of DraSSB and poly(dT) was measured with an Applied Photophysics p* system using tryptophan ﬂuores- cence excited at 295 nm and observed through a WG320 ﬁlter. Several traces collected for each concentration were accumu- lated to reduce noise. When analyzing the binding of SSB proteins to long ssDNA, a simple bimolecular model is insuf- ﬁcient, since one has to consider the length distribution of gaps between protein covered sites. At intermediate saturation, there will be gaps between bound proteins too short to accom- modate binding of further ligands. A thermodynamic equilib- rium model for such a binding of a multidentate ligand to a long linear polymer (24) has been extended to a kinetic model (10,25). In this model, it is assumed that in the rate-limiting bimolecular step of association, SSB forms an initial stable encounter complex with a shorter binding site length and subsequently redistributes in a monomolecular manner on the ssDNA. Data measured at different concentrations were analyzed by globally ﬁtting the parameters of this kinetic model using the program BPCFIT (18). RESULTS Protein sequence We ampliﬁed the postulated reading frame for D.radiodurans SSB protein from the strain DSM20539. Sequencing of the PCR product yielded an open reading frame (ORF) coding for 301 amino acids. The sequence was at variance with the genomic sequence published (EMBL accession no. AE001873, gene DR0099), which obviously contained sequencing errors leading to frameshifts and an early termination sequence. The same discrepancy was also found recently by others (9). The corrected DraSSB sequence was deposited in the EMBL database [accession numbers AJ564860 (this paper) and AY293617 (9)]. The ORF shows a large sequence similarity to other SSB proteins, such as SSBs from Thermus aquaticus