the protein itself and its protein–protein and protein–DNA interactions. As in the structure, the overall properties of DraSSB resemble those of the homotetrameric SSBs albeit with some differences in detail. MATERIALS AND METHODS Buffers and reagents D.radiodurans R1 strain DSM20539 was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany). Poly(dT) ( 1400 nt in length), poly(rU) and poly(dA–dT) were purchased from Amersham Biosciences. Polynucleotide concentrations are given in monomer residues throughout the text and were determined spectrophotometri- cally using absorption coefﬁcients of 8600 M 1 cm 1 for poly(dT) at maximum (10) and 9200 M 1 cm 1 and 6700 M 1 cm 1 at 260 nm for poly(rU) and poly(dA–dT), respectively (11). Protein concentrations were determined using the absorp- tion coefﬁcients at 280 nm calculated from amino acid com- position (12): 29 400 M 1 cm 1 for c subunit of E.coli DNA polymerase III, 82 000 M 1 cm 1 for DraSSB dimer and 113 000 M 1 cm 1 for EcoSSB tetramer. DraSSB and EcoSSB concentrations are given in dimers and tetramers, respectively, throughout the text. All experiments were carried out in potassium phosphate buffer at pH 7.4 (KPi) containing NaCl as neutral salt. Con- centrations are given at the respective experiments. Cloning of D.radiodurans ssb gene The ssb gene was ampliﬁed from heat disrupted D.radiodurans cells by PCR. Primers used were 50-GGAGACCATGGCCC- GAGGCATGAACCA-30 for forward and 50-GAAGAGGAT- CCTCATGT TGGGTGTCCT TGGTG-30 for reverse priming. Primers were selected to include start and stop codon contain- ing sequences (italic) of the DraSSB gene accession no. AJ564860 (vide infra). PCR was carried out using Pfu DNA polymerase (Stratagene), 200 mM dNTP and 0.5 mM of each primer with an annealing temperature of 53 C and 30 cycles. The PCR product was puriﬁed using a PCR puri- ﬁcation kit (Qiagen). The ampliﬁed product was digested with NcoI and BamHI and cloned into the NcoI and BamHI sites of pET15b (Novagen) to generate pET15b-DraSSB. This plasmid was used to transform the E.coli strain LK111l. The procedure was repeated using a different lot of the D.radiodurans strain DSM20539. Different clones were selec- ted and the DraSSB gene was completely sequenced in both strands (MWG Biotech) for both lots of D.radiodurans used without ﬁnding any differences. Expression and purification of SSB protein from D.radiodurans For protein expression, the plasmid pET15b-DraSSB was used to transform the E.coli strain BL21(DE3) pLysS. A volume of 900 ml of an overnight culture grown in the presence of 100 mg/ml ampicillin and 30 mg/ml chloramphenicol at 30 C was used to inoculate 10 l of standard Luria–Bertani (LB) medium and the cells were grown at 30 C. An aliquot of 100 ml of the overnight culture was used for plasmid preparation (Qiagen) and subsequent DNA sequencing to Nucleic Acids Research, 2005, Vol. 33, No. 5 1663 conﬁrm the sequence of the expressed gene shortly before induction. Protein expression was induced after growing the cells to A600 nm = 1.0 by the addition of 1 mM isopropyl-b-D- thiogalactopyranoside and was continued for 4 h. After cell harvesting and washing with buffer W [0.2 M NaCl, 50 mM Tris–HCl, pH 8.3, 1 mM EDTA and 10% (w/v) sucrose], the pellet was resuspended in buffer S (buffer W containing 15 mM spermidin), frozen and stored at 70 C. When cells were thawed, one tablet of ‘complete EDTA- free’ protease inhibitor (Roche) per 50 ml and 0.04% sodium- desoxycholat were added. After soniﬁcation and removing of cell debris by preparative ultracentrifugation, proteins were precipitated by the addition of polyethylenimine, pH 6.9 (ﬁnal concentration 0.4%) for 20 min at 4 C and pelleted. The pellet was extracted with TGE400 buffer [0.4 M NaCl, 50 mM Tris–HCl, pH 8.3, 1 mM EDTA and 20% (v/v) gly- cerol] and insoluble material was removed by centrifugation. The clear supernatant was precipitated with 20% (w/v) (NH4)2SO4 overnight at 4 C. The pellet was redissolved in TGE400 and loaded on a BlueSepharose column (13) equi- librated in the same buffer. After washing the column with TGE400, a linear salt gradient (0.4–3 M NaCl in the same buffer) was used to elute the protein. Fractions were analyzed by SDS–PAGE (14), pooled and precipitated with 20% (w/v) (NH4)2SO4. The pellet was redissolved in SBP buffer [0.5 M NaCl, 20 mM KPi, pH 7.4, 1 mM EDTA and 20% (v/v) glycerol] and dialyzed against the same buffer containing 1 M NaCl and 60% (v/v) glycerol and stored at 20 C. The protein preparation was at least 97% pure as judged from Coomassie Brillant Blue stained SDS gel. Complementation assay In E.coli strain RDP268, the chromosomal SSB gene is replaced by a kanamycin resistance and the essential SSB protein is encoded by the plasmid pACYCssb (15). This plasmid confers chloramphenicol resistance to the cells. pACYCssb is essential for the survival of the cells and can be replaced by another plasmid, only if it contains a gene whose product can take over EcoSSB function in vivo. We used the strain RDP268 to transform the pSF1 (16) derivative pSF1-DraSSB, which carries the DraSSB gene under the con- trol of the l pL promotor and confers ampicillin resistance. In RDP268 (pSF1-DraSSB), the pL promotor is not repressed leading to a high constitutive DraSSB expression. The cells were grown in the presence of 100 mg/ml ampicillin and 5 mg/ml kanamycin but omitting chloramphenicol. After four subsequent inoculations of 4 ml LB medium, allow- ing the cells to grow for 24 h at 37 C, 100–200 cells were plated on LB medium containing 100 mg/ml ampicillin and 5 mg/ml kanamycin. Clones that lost the plasmid pACYCssb were identiﬁed by replica plating on LB medium containing 30 mg/ml chloramphenicol and 5 mg/ml kanamycin. SSB pro- teins in chloramphenicol-sensitive clones were characterized by western blot analysis. Western blot analysis SSB proteins blotted onto a PVDF membrane were detected immunologically by a polyclonal anti-EcoSSB serum from rabbit. Rabbit antibodies were marked by alkaline phosphatase- conjugated goat anti-rabbit antibodies and detected by alkaline