Displacement of the canonical single-stranded DNA-binding protein in the Thermoproteales Sonia Paytubia,1,2, Stephen A. McMahona,2, Shirley Grahama, Huanting Liua, Catherine H. Bottinga, Kira S. Makarovab, Eugene V. Kooninb, James H. Naismitha,3, and Malcolm F. Whitea,3 aBiomedical Sciences Research Complex, University of St Andrews, Fife KY16 9ST, United Kingdom; and bNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 AUTHOR SUMMARY Proteins are the major structural and biochemical route involved direct puri- operational components of cells. Even fication and identification of proteins the simplest organisms possess hundreds thatcouldbindtossDNAinoneofthe of different proteins, and more complex Thermoproteales, Thermoproteus tenax. organisms typically have many thou- This approach resulted in the identifi- sands. Because all living beings, from cation of the product of the gene ttx1576 microbes to humans, are related by as a candidate for the missing SSB. evolution, they share a core set of pro- We proceeded to characterize the teins in common. Proteins perform properties of the Ttx1576 protein, (which fundamental roles in key metabolic we renamed “ThermoDBP” for Ther- processes and in the processing of in- moproteales DNA-binding protein), formation from DNA via RNA to pro- showing that it has all the biochemical teins. A notable example is the ssDNA- properties consistent with a role as a binding protein, SSB, which is essential functional SSB, including a clear prefer- for DNA replication and repair and is ence for ssDNA binding and low se- widely considered to be one of the quence specificity. Using crystallographic few core universal proteins shared by analysis, we solved the structure of the alllifeforms.Herewedemonstrate Fig. P1. A schematic representation of the tree DNA-binding domain of ThermoDBP, that one branch of the tree of life has of life with the domains Eukarya, Bacteria, and revealing a protein fold with a promi- lost this “ubiquitous” protein and Archaea indicated. The archaeal domain is nent cleft punctuated with aromatic expanded to highlight the different subdivisions replaced it with another, unrelated one. amino acid residues and lined by posi- fi and to highlight the Thermoproteales, which are This nding has important implications boxed. All forms of life use the canonical ssDNA- tively charged residues. The structure of for our understanding of the plasticity binding protein based on the OB-fold, which is ThermoDBP immediately suggested of evolution. represented in cyan, with the exception of the a mechanism for the binding of ssDNA Rapid advances in genome-sequencing Thermoproteales, which use the completely along the cleft that is reminiscent of technology over the past 15 y yielded unrelated protein ThermoDBP for ssDNA binding. the binding clefts of canonical SSB a wealth of new information on many proteins but is unrelated to them in se- divergent parts of the tree of life that can quence and detailed structure. The two be mined for information to illuminate all aspects of biology. The ssDNA-binding domains are linked by a C-terminal helical tree of life consists of three fundamental divisions known as coiled-coil domain that allows ThermoDBP to dimerize “domains”: Eukarya (organisms with a nucleus where DNA is (Fig. P1). stored, including plants, fungi, animals) and the prokaryotic In conclusion, we used biochemistry and bioinformatics to Bacteria and Archaea, which lack a nucleus (1). One of the most demonstrate the displacement of an essential, “universal” pro- highly conserved proteins found in all three domains is the SSB tein by a completely unrelated one in one branch of the tree of protein, which binds and protects ssDNA during replication and life. The structure of ThermoDBP reveals a unique solution to repair of damage to the genome. It is an essential protein that the problem of ssDNA binding. This result suggests that even is thought to have been present in the last common ancestor the most fundamental, ubiquitous proteins can be replaced of all extant life (2). The defining feature of all known SSBs is during evolution. the oligonucleotide-binding (OB) fold shown in Fig. P1. Recently we noted that one group of archaeal species, the Thermopro- teales, lack a detectable gene for the SSB protein in their Author contributions: J.H.N. and M.F.W. designed research; S.P., S.A.M., S.G., H.L., C.H.B., genomes (3). and K.S.M. performed research; S.P., S.A.M., C.H.B., E.V.K., J.H.N., and M.F.W. analyzed Because a functional SSB is likely to be essential for any data; and S.P., S.A.M., K.S.M., E.V.K., J.H.N., and M.F.W. wrote the paper. organism, we reasoned that the Thermoproteales might have The authors declare no conflict of interest. lost the canonical SSB gene and replaced it with another, un- This article is a PNAS Direct Submission. related gene. To test this hypothesis, we undertook a two- Data deposition: The atomic coordinates and structure factors have been deposited in the pronged approach comprising a combination of bioinformatics Protein Data Bank database, www.pdb.org (PDB ID code 3TEK). and biochemistry. The bioinformatic analysis involved a search 1Present address: Departament de Microbiologia, Facultat de Biologia, Universitat de for any genes that were common to the 10 Thermoproteales Barcelona, 08028 Barcelona, Spain. species lacking the canonical SSB and that were not found in 2S.P. and S.A.M. contributed equally to this work. any other genome. Remarkably, only a single gene fits these 3To whom correspondence may be addressed. E-mail: [email protected] or mfw2@ criteria, ttx1576. This observation is clearly compatible with st-and.ac.uk. the possibility that the Ttx1576 protein compensates function- See full research article on page E398 of www.pnas.org. ally for the missing SSB protein in Thermoproteales. The Cite this Author Summary as: PNAS 10.1073/pnas.1113277108. 2198–2199 | PNAS | February 14, 2012 | vol. 109 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1113277108 Downloaded by guest on September 30, 2021 1. Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: 2. Mushegian AR, Koonin EV (1996) A minimal gene set for cellular life derived by PNAS PLUS proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 87: comparison of complete bacterial genomes. Proc Natl Acad Sci USA 93:10268–10273. 4576–4579. 3. Luo X, et al. (2007) CC1, a novel crenarchaeal DNA binding protein. J Bacteriol 189: 403–409. EVOLUTION Paytubi et al. PNAS | February 14, 2012 | vol. 109 | no. 7 | 2199 Downloaded by guest on September 30, 2021.