Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage

Abstract

Single-stranded DNA (ssDNA)-binding (SSB) proteins are uniformly required to bind and protect single-stranded intermediates in DNA metabolic pathways. All bacterial and eukaryotic SSB proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide͞oligosaccharide-binding (OB) fold, that cooperate in nonspecific ssDNA binding. The vast majority of bacterial SSB family members function as homotetramers, with each monomer contributing a single OB fold. However, SSB proteins from the Deinococcus-Thermus genera are exceptions to this rule, because they contain two OB folds per monomer. To investigate the structural consequences of this unusual arrangement, we have determined a 1.8-Å-resolution x-ray structure of Deinococcus radiodurans SSB. The structure shows that D. radiodurans SSB comprises two OB domains linked by a ␤-hairpin motif. The protein assembles a four-OB-fold arrangement by means of symmetric dimerization. In contrast to homotetrameric SSB proteins, asymmetry exists between the two OB folds of D. radiodurans SSB because of sequence differences between the domains. These differences appear to reflect specialized roles that have evolved for each domain. Extensive crystallographic contacts link D. radiodurans SSB dimers in an arrangement that has important implications for higher-order structures of the protein bound to ssDNA. This assembly utilizes the N-terminal OB domain and the ␤-hairpin structure that is unique to Deinococcus and Thermus species SSB proteins. We hypothesize that differences between D. radiodurans SSB and homotetrameric bacterial SSB proteins may confer a selective advantage to D. radiodurans cells that aids viability in environments that challenge genomic stability.I n all organisms, single-stranded DNA (ssDNA)-binding (SSB) proteins sequester and protect ssDNA intermediates that arise during DNA replication, recombination, and repair (1). Their prominent roles in genome maintenance reactions make SSB proteins a requirement for cellular life (2). Although the sequences of SSB family members are highly variable, two common functional themes have emerged that link this class of proteins across evolution. The first is that SSB proteins use a conserved domain called an oligonucleotide͞oligosaccharide-binding (OB) fold to bind ssDNA (3, 4). OB domains bind ssDNA in a cleft formed primarily by ␤-strands, by using aromatic residues that stack against nucleotide bases and positively charged residues that form ionic interactions with the DNA backbone (5-8). The second common feature of cellular SSB proteins is obligate oligomerization that brings together four DNA-binding OB folds. For example, Escherichia coli SSB contains a single OB fold per monomer, but the active form of the protein is a homotetramer with four OB folds (1). This general arrangement appears to define a structural paradigm for bacterial SSB family proteins because all but three of the Ͼ250 currently identifiable bacterial ssb genes encode proteins with a single OB fold.Different...

Structure and Cellular Dynamics of Deinococcus radiodurans Single-stranded DNA (ssDNA)-binding Protein (SSB)-DNA Complexes

“…Because the homodimeric D/T group SSBs have non-identical OB domains present in each monomer, it has been suggested that the DNA binding properties of each domain may differ for this group of SSBs (15). A crystallographic approach was taken to determine the ssDNA-bound structure of DrSSB, the founding member of the D/T group homodimeric SSBs.…”

“…Dimerization places the four OB domains in very similar positions to that found in homotetrameric SSBs, which could indicate that both classes of bacterial SSBs bind ssDNA in a conserved manner. However, because the OB domains within D/T SSB monomers are not identical in sequence, the N-and C-terminal OB domains could bind ssDNA differently (15). Structural data mapping the ssDNA binding sites of a number of homotetrameric SSBs are available (19 -22), but high resolution structures of D/T SSBs in complex with ssDNA have not been reported.…”

“…The second group, which includes SSBs found only in bacteria within the Deinococcus/ Thermus (D/T) phylum, is composed of homodimeric SSBs with two OB domains per subunit. Structural analyses of D/T SSBs from D. radiodurans and Thermus aquaticus have revealed an arrangement in which the two OB domains abut one another and are directly connected by a ␤-hairpin within each monomer (15,17,18). Dimerization places the four OB domains in very similar positions to that found in homotetrameric SSBs, which could indicate that both classes of bacterial SSBs bind ssDNA in a conserved manner.…”

Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

“…The structures of several prokaryotic ssDNA-binding proteins have been solved (4)(5)(6)(7)(8)(9). Although these proteins do not have sequence homology, their structures share a common oligosaccharide/oligonucleotide-binding fold (OB-fold), suggesting that they have a similar mode of action.…”

Crystal structure of a novel single‐stranded DNA binding protein from Mycoplasma pneumoniae

“…Approximately the first 120 N-terminal residues comprise the OB/SSB fold with conserved structure whereas the rest of the protein has been implicated in interacting with other protein partners and in some cases has been disordered in the crystal structures. Numerous prior efforts have reported in detail the crystal structures of functionally annotated SSBs, [12][13][14][15][16][17][18][19][20][21][22][23] presented comparative analysis of homologous SSBs 20,24 and reported on structures of their protein-DNA complexes. 25,26 An electrophoretic mobility shift assay confirmed that MPN554 binds to a 39-mer single-stranded DNA.…”