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

Single-stranded DNA binding proteins are essential in nearly all organisms and play central roles in all aspects of genome maintenance, including DNA replication, recombination and repair (1–3). They bind selectively single stranded DNA with high affinity and independent of sequence specificity, protecting ssDNA intermediates from degradation and preventing them from the formation of secondary structures. The other functions of SSB proteins have become recognized only recently and include the regulation of the activity of many other DNA metabolic proteins through direct protein-protein interactions (4) as well as by modulating the accessibility of ssDNA by either different modes of binding (3, 5) or by the ability to translocate along ssDNA as shown recently for EcoSSB (6). Although SSB proteins from different organisms can differ in architecture, for example, the bacteriophage T4 gene 32 protein is a monomer (7, 8), EcoSSB protein is a homotetramer (9–11) and eucaryotic RPA proteins are heterotrimers (12, 13), they all contain oligonucleotide/oligosaccharide binding folds (OB-fold), which form the primary site for ssDNA binding (14, 15). The number of OB folds varies significantly in these proteins, from one in T4 gene 32 protein to six in the hetero-trimeric RPAs. Most of the bacterial SSBs are homotetramers and contain 4 OB-folds (one per monomer) (see for example (16–19), with the EcoSSB tetramer being the most studied. In contrast to the homotetrameric SSB proteins, SSB proteins from Deinococcus-Thermus group function as homodimers; however, each monomer encodes two OB-folds linked by a conserved spacer sequence (20–23). Therefore, the functional form of these proteins still is composed of four OB-folds, although the sequence differences between the two OB-folds within each dimer impose an asymmetry that is likely to affect its DNA binding properties. Furthermore, these dimers possess only two unstructured C-termini, rather than four as is the case for the homo-tetrameric SSB proteins. DrSSB protein originates from one of the most radiation resistant organisms known (24). D. radiodurans, which is a soil bacterium featuring a D37 γ irradiation dose of approximately 6,000 Gy, making this organism roughly 200 times more radiation resistant than Escherichia coli (24). DrSSB forms a stable homodimer in solution (22) and its quantitative estimates in the D. radiodurans cell indicate approximately 2500–3000 dimers/cell, independent of the level of irradiation (25). A crystal structure of the full length DrSSB was determined with 1.8A resolution (23) and shows that each DrSSB subunit comprises two OB-folds linked by a β-hairpin motif and the protein assembles a four-OB-fold arrangement by means of symmetric dimerization. The C-terminal tail residues 234–301 are missing from the model due to the lack of electron density suggesting that the extended C-terminal region of this protein is highly unstructured similar to the C-terminal tails of EcoSSB (11). In order to better understand the functional consequences arising from these differences, we have examined the binding of Deinococcus radiodurans SSB (DrSSB) to a variety of ssDNA to compare its DNA binding properties with those of the well-studied homo-tetrameric EcoSSB.