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2007 | 54 | 1 | 79-87
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Identification and properties of the Deinococcus grandis and Deinococcus proteolyticus single-stranded DNA binding proteins (SSB)

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To study the biochemical properties of SSB's from Deinococcus grandis (DgrSSB) and Deinococcus proteolyticus (DprSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli overexpression systems. The genes consist of an open reading frame of 891 (DgrSSB) and 876 (DprSSB) nucleotides encoding proteins of 296 and 291 amino acids with a calculated molecular mass of 32.29 and 31.33 kDa, respectively. The amino-acid sequence of DgrSSB exhibits 45%, 44% and 82% identity and the amino-acid sequence of DprSSB reveals 43%, 43% and 69% identity with Thermus aquaticus (TaqSSB), Thermus thermophilus (TthSSB) and Deinococcus radiodurans SSBs, respectively. We show that DgrSSB and DprSSB are similar to Thermus/Deinococcus SSBs in their biochemical properties. They are functional as homodimers, with each monomer encoding two single-stranded DNA binding domains (OB-folds). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 33 nt per homodimer. In a complementation assay in E. coli, DgrSSB and DprSSB took over the in vivo function of EcoSSB. Thermostability with half-lives of about 1 min at 65-67.5°C make DgrSSB and DprSSB similar to the known SSB of Deinococcus radiodurans (DraSSB).
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  • Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, Gdańsk, Poland
  • Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, Gdańsk, Poland
  • Bernstein DA, Eggington JM, Killoran MP, Misic AM, Cox MM, Keck JL (2004) Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage. Proc Natl Acad Sci USA 101: 8575-8580.
  • Bochkarev A, Pfuetzner RA, Edwards AM, Frappier L (1997) Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature 385: 176-181.
  • Bochkarev A, Bochkareva E, Frappier L, Edwards AM (1999) The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J 18: 4498-4504.
  • Curth U, Genschel J, Urbanke C, Greipel J (1996) In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein. Nucleic Acids Res 24: 2706-2711.
  • Dabrowski S, Kur J (1999) Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification. Protein Expr Purif 16: 96-102.
  • Dabrowski S, Olszewski M, Piatek R, Brillowska-Dabrowska A, Konopa G, Kur J (2002a) Identification and characterization of single-stranded-DNA-binding proteins from Thermus thermophilus and Thermus aquaticus - new arrangement of binding domains. Microbiology 148: 3307-3315.
  • Dabrowski S, Olszewski M, Piatek R, Kur J (2002b) Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus - expression and purification. Protein Expr Purif 26: 131-138.
  • de Groot A, Chapon V, Servant P, Christen R, Saux MF, Sommer S, Heulin T (2005) Deinococcus deserti sp. nov., a gamma-radiation-tolerant bacterium isolated from the Sahara Desert. Int J Syst Evol Microbiol 55: 2441-2446.
  • Eggington JM, Haruta N, Wood EA, Cox MM (2004) The single-stranded DNA-binding protein of Deinococcus radiodurans. BMC Microbiol 4: 2.
  • Ferreira AC, Nobre MF, Rainey FA, Silva MT, Wait R, Burghardt J, Chung AP, da Costa MS (1997) Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs. Int J Syst Bacteriol 47: 939-947.
  • Filipkowski P, Duraj-Thatte A, Kur J (2006a) Novel thermostable single-stranded DNA-binding protein (SSB) from Deinococcus geothermalis. Arch Microbiol 186: 129-137.
  • Filipkowski P, Koziatek M, Kur J (2006b) A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans. Extremophiles 10: 607-614.
  • Genschel J, Curth U, Urbanke C (2000) Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease. Biol Chem 381: 183-192.
  • Handa P, Acharya N, Varshney U (2001) Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases. J Biol Chem 276: 16992-16997.
  • Hirsch P, Gallikowski CA, Siebert J, Peissl K, Kroppenstedt R, Schumann P, Stackebrandt E, Anderson R (2004) Deinococcus frigens sp. nov., Deinococcus saxicola sp. nov., and Deinococcus marmoris sp. nov., low temperature and draught-tolerating, UV-resistant bacteria from continental Antarctica. Syst Appl Microbiol 27: 636-645.
  • Jedrzejczak R, Dauter M, Dauter Z, Olszewski M, Dlugolecka A, Kur J (2006) Structure of the single-stranded DNA-binding protein SSB from Thermus aquaticus. Acta Crystallogr D Biol Crystallogr 62: 1407-1412.
  • Kelman Z, Yuzhakov A, Andjelkovic J, O'Donnell M (1998) Devoted to the lagging strand - the χ-subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly. EMBO J 17: 2436-2449.
  • Kur J, Olszewski M, Dlugolecka A, Filipkowski P (2005) Single-stranded DNA-binding proteins (SSBs) - sources and applications in molecular biology. Acta Biochim Polon 52: 569-574.
  • Lohman TM, Overman LB (1985) Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration. J Biol Chem 260: 3594-3603.
  • Murzin AG (1993) OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12: 861-867.
  • Omelchenko MV, Wolf YI, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, Daly MJ, Koonin EV, Makarova KS (2005) Comparative genomics of Thermus thermophilus and Deinococcus radiodurans: divergent routes of adaptation to thermophily and radiation resistance. BMC Evol Biol 5: 57.
  • Porter RD, Black S (1991) The single-stranded-DNA-binding protein encoded by the Escherichia coli F factor can complement a deletion of the chromosomal ssb gene. J Bacteriol 173: 2720-2723.
  • Porter RD, Black S, Pannuri S, Carlson A (1990) Use of the Escherichia coli SSB gene to prevent bioreactor takeover by plasmidless cells. Biotechnology (N Y) 8: 47-51.
  • Raghunathan S, Kozlov AG, Lohman TM, Waksman G (2000) Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat Struct Biol 7: 648-652.
  • Shamoo Y, Friedman AM, Parsons MR, Konigsberg WH, Steitz TA (1995) Crystal structure of a replication fork single-stranded DNA binding protein (T4 gp32) complexed to DNA. Nature 376: 362-366.
  • Stassen MJ, Bailey D, Nelson S, Chinwalla V, Harte PJ (1995) The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins. Mech Dev 52: 209-223.
  • Wadsworth RI, White MF (2001) Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus. Nucleic Acids Res 29: 914-920.
  • Webster G, Genschel J, Curth U, Urbanke C, Kang C, Hilgenfeld R (1997) A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria. FEBS Lett 411: 313-316.
  • Williams KR, Konigsberg W (1978) Structural changes in the T4 gene 32 protein induced by DNA polynucleotides. J Biol Chem 253: 2463-2470.
  • Witte G, Urbanke C, Curth U (2003) DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery. Nucleic Acids Res 31: 4434-4440.
  • Witte G, Urbanke C, Curth U (2005) Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization. Nucleic Acids Res 33: 1662-7160.
  • Yang C, Curth U, Urbanke C, Kang C (1997) Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 A resolution. Nat Struct Biol 4: 153-157.
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