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Identification and characterization of single-stranded-DNA-binding proteins from Thermus thermophilus and Thermus aquaticus – new arrangement of binding domains^b

Sawomir Dbrowski¹

Rafa Pitek¹

Anna Brillowska-Dbrowska¹

Grayna Konopa²

Technical University of Gdansk, Department of Microbiology, ul. G. Narutowicza 11/12, 80-952 Gdansk, Poland¹
Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland²

Author for correspondence: Józef Kur. Tel: +48 58 3471822. Fax: +48 58 3471822. e-mail: kur{at}altis.chem.pg.gda.pl

Single-stranded-DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in bacteria, archaea and eukarya. This paper reports the identification and characterization of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and Thermus aquaticus. These proteins (TthSSB and TaqSSB), in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers, and each monomer contains two ssDNA-binding domains with a conserved OB (oligonucleotide/oligosaccharide-binding) fold, as deduced from the sequence analysis. The N-terminal domain is located in the region from amino acid 1 to 123 and the C-terminal domain is located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively. Purified TthSSB or TaqSSB binds only to ssDNA and with high affinity. The binding site size for TaqSSB and TthSSB protein corresponds to 30–35 nucleotides. It is concluded that the SSBs of thermophilic and mesophilic bacteria, archaea and eukarya share a common core ssDNA-binding domain. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life.

Keywords: DNA replication, expression, purification, thermophilic bacteria, thermostability

Abbreviations: dsDNA, double-stranded DNA; OB fold, oligonucleotide/oligosaccharide-binding fold; RPA, replication protein A; SSB, single-stranded-DNA-binding protein (EcoSSB, Escherichia coli SSB; HsmtSSB, human mitochondrial SSB; TaqSSB, Thermus aquaticus SSB; TthSSB, Thermus thermophilus SSB); ssDNA, single-stranded DNA

^b The GenBank accession numbers for the sequences reported in this paper are AF079160 and AF276705.

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