Binding of the dimeric Deinococcus radiodurans single-stranded DNA binding protein to single-stranded DNA.

Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) originates from a radiation-resistant bacterium and participates in DNA recombination, replication, and repair. Although it functions as a homodimer, it contains four DNA binding domains (OB-folds) and thus is structurally similar to the Escherichia coli SSB (EcoSSB) homotetramer. We examined the equilibrium binding of DrSSB to ssDNA for comparison with that of EcoSSB. We find that the occluded site size of DrSSB on poly(dT) is ∼45 nucleotides under low-salt conditions (<0.02 M NaCl) but increases to 50-55 nucleotides at ≥0.2 M NaCl. This suggests that DrSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB, although the site size difference between modes is not as large as for EcoSSB, suggesting that the pathways of ssDNA wrapping differ for these two proteins. The occluded site size corresponds well to the contact site size (52 nucleotides) determined by isothermal titration calorimetry (ITC). Electrophoretic studies of complexes of DrSSB with phage M13 ssDNA indicate the formation of stable, highly cooperative complexes under low-salt conditions. Using ITC, we find that DrSSB binding to oligo(dT)s with lengths close to the determined site size (50-55 nucleotides) is stoichiometric with a ΔH(obs) of approximately -94 ± 4 kcal/mol, somewhat smaller than that for EcoSSB (approximately -130 kcal/mol) under the same conditions. The observed binding enthalpy shows a large sensitivity to NaCl concentration, similar to that observed for EcoSSB. With the exception of the less dramatic change in occluded site size, the behavior of DrSSB is similar to that of EcoSSB protein (although clear quantitative differences exist). These common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.