1662–1670 Nucleic Acids Research, 2005, Vol. 33, No. 5 doi:10.1093/nar/gki310 Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization Gregor Witte, Claus Urbanke and Ute Curth* Department of Biophysical Chemistry, Medical School Hannover, Hannover, Germany Received January 28, 2005; Revised and Accepted February 28, 2005 DDBJ/EMBL/GenBank accession no. AJ564860 ABSTRACT The highly conserved bacterial single-stranded DNA-binding (SSB) proteins play an important role in DNA replication, repair and recombination and are essential for the survival of the cell. They are func- tional as tetramers, in which four OB(oligonucleotide/ oligosaccharide binding)-folds act as DNA-binding domains. The protomer of the SSB protein from the extremely radiation-resistant organism Deinococcus radiodurans (DraSSB) has twice the size of the other bacterial SSB proteins and contains two OB-folds. Using analytical ultracentrifugation, we could show that DraSSB forms globular dimers with some protru- sions. These DraSSB dimers can interact with two molecules of E.coli DNA polymerase III x subunit. In fluorescence titrations with poly(dT) DraSSB bound 47–54 nt depending on the salt concentration, and fluorescence was quenched by more than 75%. A distinct low salt binding mode as for EcoSSB was not observed for DraSSB. Nucleic acid binding affin- ity, rate constant and association mechanism are quite similar for EcoSSB and DraSSB. In a comple- mentation assay in E.coli, DraSSB took over the in vivo function of EcoSSB. With DraSSB behaving almost identical to EcoSSB the question remains open as to why dimeric SSB proteins have evolved in the Thermus group of bacteria. INTRODUCTION Single-stranded DNA-binding (SSB) proteins play a vital role in the sustainment of almost every form of life we know on earth. They protect and conﬁgure the vulnerable state of unwound and single-stranded DNA (ssDNA) for efﬁcient use by enzymes involved in DNA metabolism, such as rep- lication, recombination and repair. The function of ssDNA binding is found in seemingly very different protein classes ranging from monomers over homodimers and homotetramers to heterooligomers. However, all SSB proteins share a structural motif called OB(oligonucleotide/oligosaccharide binding)-fold (1,2). One of these classes is formed by homotet- rameric SSB proteins which play a vital role in bacteria and eukaryotic mitochondria. Their 3D structures, which have been determined for such divergent organisms as E.coli and human, are quite similar (3–5), and thus this class of proteins constitutes an evolutionary strictly conserved structural principle. In light of this structural conservation, it was surprising when in silico analysis of the ssb genes from the bacterial Thermus group suggested the proteins encoded by these genes to belong to the class of homotetrameric SSBs, although they were about twice the size compared with other proteins of this class. The protomers of Thermus SSBs contain two OB-folds per monomer, and therefore it was not surprising that these proteins form homodimers (6). These homodimers thus mimic the homotetrameric SSBs. One might argue that the origin of homodimeric SSB pro- teins was a duplication of the gene of the homotetrameric SSB proteins, and there have been some speculations about evolu- tionary raison d’eˆtre for such a duplication. One of these speculations puts an evolutionary advantage to the fact that the two OB-folds in the homodimeric SSB can evolve separ- ately and thus can help in surviving the hostile environments Thermus bacteria live in (7). Recently, several homodimeric SSBs from different Thermus species have been cloned and isolated as proteins (8,9) and for the SSB from Deinococcus radiodurans (DraSSB) a 3D structure has been determined by X-ray crystallography (7). The overall structure of DraSSB in its OB-folds is quite similar to the human mitochondrial and E.coli SSB structure. However, owing to the lack of solid biochemical and biophysical data on the solution properties of DraSSB, the interpretation of structural details remains speculative. We, therefore, have isolated the gene for this protein, expressed it in E.coli and puriﬁed it to homogeneity. In this work, we will describe the biophysical and biochemical characterization of *To whom correspondence should be addressed at Medizinische Hochschule Hannover, Zentrale Einrichtung fu¨r biophysikalisch-biochemische Verfahren, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. Tel: +49 511 532 3707; Fax: +49 511 532 5966; Email: curth.ute@mh-hannover.de ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org