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

Slawomir Dabrowski1, Marcin Olszewski1, Rafal Piatek1, Anna Brillowska-Dabrowska1, Grazyna Konopa2 and Jozef Kur1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Single-stranded DNA-binding proteins (SSBs) are indispensable elements in cells of all living organisms. SSB proteins interact with ssDNA in sequence in an independent manner, preventing them from forming secondary structures and from degradation by nucleases (Greipel et al., 1989 ). In such a manner, SSB-binding proteins participate in all processes involving ssDNA, such as replication, repair and recombination (Alani et al., 1992 ; Greipel et al., 1989 ; Lohman & Overman, 1985 ; Meyer & Laine, 1990 ; Moore et al., 1991 ). SSB proteins, which are present in all three branches of living organisms and in viruses, share sequences as well as biochemical and structural characteristics.

The bacterial SSB proteins described to date work in vivo as homotetramers (for example see de Vries & Wackernagel, 1993 , 1994 ; Genschel et al., 1996 ; Purnapatre & Varshney, 1999 ). The monomer is built from two fragments: (i) the N-terminal fragment (approx. 120 amino acids possessing conserved residues that are responsible for binding to ssDNA, tetramerization and stabilization of the monomer fold (Carlini et al., 1998 ; Williams et al., 1983 ), and (ii) the C-terminal fragment, which displays a very low sequence conservation except for the few last amino acids. This C-terminal fragment is responsible for interactions of bacterial SSB proteins with other proteins. Eukaryotic human mitochondrial SSB protein (HsmtSSB), despite low sequence homology to Escherichia coli SSB (EcoSSB) (36% identity), displays similar biochemical characteristics (Curth et al., 1994 ). HsmtSSB proteins, like their counterparts from bacteria, are homotetramers (Webster et al., 1997 ). The crystal structure of HsmtSSB proteins reveals a nearly identical fold as in the structure of EcoSSB (Webster et al., 1997 ; Yang et al., 1997). Also the mode of dimerization and tetramerization of HsmtSSB and EcoSSB is almost identical (Webster et al., 1997 ; Yang et al., 1997 ). Residues critical for interaction of HsmtSSB protein with ssDNA are also homologous to conserved residues in EcoSSB (Webster et al., 1997 ; Yang et al., 1997 ). Inspection of the structures of HsmtSSB and EcoSSB shows that binding to ssDNA in both proteins is governed by positively charged concavities located on the surface (Matsumoto et al., 2000 ; Raghunathan et al., 2000 ; Yang et al., 1997 ).

Replication protein A (RPA) has been identified as a eukaryotic nuclear ssDNA-binding protein (Smith et al., 1997 ). RPA is a heterotrimer composed of subunits of 70, 32 and 14 kDa, each of which is conserved in all eukaryotes (Brill & Stillman, 1991 ). Biochemical and also structural data show that RPA70 possesses two ssDNA-binding domains; RPA32 and RPA14 possess only one ssDNA-binding domain (Bochkarev et al., 1997 , 1999 ; Gomes & Wold, 1995 , 1996 ; Pfuetzner et al., 1997 ). The whole RPA protein possesses four ssDNA-binding domains which share structural similarities (Bochkarev et al., 1999 ). A special conserved feature of the homologies of the RPA70 protein not observed in prokaryotic SSBs is the presence of a C-4 zinc-finger motif in the C-termini, which is required for effective participation of RPA proteins in such processes as DNA replication and mismatch repair (Bochkareva et al., 2000 ; Lin et al., 1998 ).

Kelly et al. (1998) identified the first archaeal SSB protein in the genome sequence of Methanococcus jannaschii. In searching for putative ORFs for SSB proteins in Archaea, they used a known sequence of human protein RPA70 and identified an M. jannaschii SSB protein that is 645 amino acids long and exists in solution as a monomer. M. jannaschii SSB protein is built from four tandem repeats of the common core ssDNA-binding domain (Kelly et al., 1998 ). In the sequence of another methanogen, Methanobacterium thermoautrophicum, a homologue of eukaryotic RPA70 protein was identified which possesses five putative consecutive repeats of the OB (oligonucleotide/oligosaccharide binding) fold (Kelman et al., 1999 ). In the M. jannaschii and M. thermoautrophicum sequences, one putative zinc finger motif was detected.

Recently the RPA homologue in Pyrococcus furiosus was cloned and characterized (Komori & Ishino, 2001 ). As in eukaryotes, the functional P. furiosus RPA protein is a heterotrimer consisting of three subunits: RPA41, RPA32 and RPA14. RPA41 is homologous to RPA70; it possesses two OB folds and a characteristic zinc-finger motif (RPA32 and RPA14 possess one putative OB fold). Sequence alignment showed that the zinc-finger motif is conserved between eukaryotic RPA70 proteins and their archaeal homologues.

Analysis of all known structures and sequences of SSB proteins from prokaryotes, archaea and eukaryotes shows that they possess a common ancestor. The ancestor SSB protein probably possessed four ssDNA-binding domains with the features seen in the core of all currently known SSB proteins with an OB fold (Murzin, 1993 ).

Here, we present first the biochemical characterization of two SSB-like proteins from the thermophilic bacteria Thermus thermophilus and Thermus aquaticus, TthSSB and TaqSSB. The results show that these proteins are homodimers, in contrast to their counterparts from mesophilic bacteria, archaea and eukarya. Close sequence analysis displays that each TthSSB or TaqSSB monomer contains two ssDNA-binding domains with a conserved OB fold. We speculate that the fusion of two ssDNA-binding domains in one polypeptide is an adaptation of thermophilic bacteria to extreme conditions.


   METHODS
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METHODS
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DISCUSSION
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Expression and purification of the recombinant TthSSB and TaqSSB.
T. thermophilus HB-8 and T. aquaticus YT-1 were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany).


Based on the known ssb-like gene sequence (GenBank accession no. AF146075) from T. thermophilus VK-1, specific primers for PCR amplification were designed and synthesized. The forward primer was ssbBgl, 5'-GCG AGA TCT TCA TAT GGC TCG AGG CCT GAA CCG CG-3' (35 nt, containing BglII and NdeI recognition sites), and the reverse primer was ssbHind, 5'-GCG AAG CTT AAA ACG GCA AAT CCT CCT CCG GCG G-3' (34 nt, containing a HindIII recognition site and a UAA stop codon, italized). The boldface parts of the primer sequences are complementary to the nucleotide sequences of the ssb-like gene of T. thermophilus VK-1; the 5' overhanging ends of the primers contain recognition sites for restriction endonucleases (underlined), and are designed to facilitate cloning. The ssb-like gene sequences of both T. thermophilus and T. aquaticus were amplified by PCR (using hyperthermostable Pwo DNA polymerase, DNA-Gdansk II, Poland) and were inserted between the BglII and HindIII sites of the bacterial expression vector pET-30LIC (Novagen). Clones were selected and sequenced to determine the nucleotide sequences of the ssb-like genes of T. thermophilus and T. aquaticus (GenBank accession nos AF079160 and AF276705, respectively). One plasmid from each cloning was selected, and used for expression and purification procedure. Transformants of E. coli BL21(DE3)pLysS were grown at 25  °C in Luria–Bertani medium to an OD600 of 0·3 and were induced by incubation in the presence of IPTG for 12 h. The purification procedure was analogous to the previously published purification scheme for the SSB from calf thymus (Atrazhev et al., 1992 ), with some modifications. The main advantage of the purification of thermostable SSB-like proteins is the reduction of contamination by the host proteins after heat treatment in the first step of purification.

Protein sequence analysis.
The amino acid sequences of TaqSSB and TthSSB were analysed using standard protein–protein BLAST and RPS-BLAST. Multiple sequence alignment was generated by using the program ClustalX. The results were prepared using the editor program Gendoc (copyright Karl Nicholas).

Estimation of the native molecular mass.
The molecular mass of TthSSB and TaqSSB proteins was determined by three independent methods: (i) gel filtration on a Superdex 200 HR 10/30 FPLC column (Pharmacia Biotech) (Siegel & Monty, 1966 ), (ii) analytical ultracentrifugation (Beckman XLA) and (iii) optimized chemical cross-linking experiments using 0·1% (v/v) glutaraldehyde for 1–20 min with TthSSB or TaqSSB concentrations between 50 and 500 µg ml-1 (Rudolf et al., 1996 ). The Gel Filtration LMW Calibration Kit (Amersham Pharmacia Biotech) was used as a calibration standard in the gel filtration method. Catalase (11S), lactate dehydrogenase (7·3S), bovine albumin (4·8S) and ovalbumin (3·5S) were loaded onto parallel tubes to serve as sedimentation markers.

Gel mobility shift assays: binding to ss oligonucleotides.
Oligonucleotides were 5'-end-labelled by fluorescein. Binding reactions were incubated for 5 min at 25 °C in buffer (20 mM Tris/HCl pH 7·5, 100 mM NaCl, 1 mM EDTA), and the reaction products were resolved by electrophoresis in 2% agarose gels (Kur et al., 1989 ).

Melting point depression of dsDNA.
Melting point curves were obtained by measuring the change in A260 in a Cary300Bio UV-Visible spectrophotometer (Varian) in buffer A2 containing 0·1 M NaCl, as described by Augustyns et al. (1991) , except for the heating rate of 20 °C h-1. The heating and cooling curves were similar. The assay was performed using duplex DNA (44 bp) composed of two oligonucleotides: 5'-G A A C C G G A G G A A T G A T G A T G A T G A T G G T G C G GTTTGTCGGACGG-3' and 5'-CCGTCCGACAAACCGCACCATCATCATCATCATTCCTCCGGTTC-3'.

Fluorescence titration.
Fluorescence titrations were carried out in a Perkin-Elmer LS-5B luminescence spectrometer as described earlier (Dabrowski & Kur, 1999 ). The binding reaction was assembled in 2 ml buffer BB (20 mM Tris/HCl pH 7·5, 1 mM EDTA) containing 0·002 or 0·1 M NaCl and was incubated at 25 and 60 °C. A constant amount of TthSSB or TaqSSB protein was incubated with varying quantities of (dT)75 oligonucleotide (Sigma). The excitation and emission wavelengths were 295 and 348 nm, respectively. Stopped-flow experiments were performed at 25 °C in a Perkin-Elmer LS-5B luminescence spectrometer and were evaluated using a model for irreversible binding of a multidentate ligand to linear polymer (Urbanke & Schaper, 1990 ). Binding of TaqSSB and TthSSB to (dT)60 and (dT)75 oligonucleotides was assayed.

Thermostability.
Thermostability assays were performed at 80, 85, 90 and 95 °C for 0, 1, 5, 10, 15, 30 and 45 min with and without 5' fluorescein-labelled oligonucleotide (dT)30 in buffer A2 containing 0·1 M NaCl. The ssDNA-binding activity was determined by gel mobility shift assay in 2% agarose gel and determination of shifted band fluorescence. Differential scanning microcalorimetry (DSC) was carried out with a power-compensated Pyris 1 DSC calorimeter (Perkin Elmer). Samples containing 1·5 mg ml-1 TaqSSB or TthSSB in 50 mM potassium phosphate buffer pH 7·5, 0·1 M NaCl were analysed. The calorimetric scans were carried out between 20 and 99 °C with a scan rate of 1 °C min-1. Each sample was scanned a second time after the actual calorimetric scan to estimate the reversibility of the unfolding transition.

Electron microscopy.
Samples of poly(dT)700 (ICN) and TaqSSB protein were incubated in TE buffer with 50 mM NaCl and 10 mM MgCl2 on ice for 20 min. Lambda phage dsDNA 1212 bp fragments (corresponding to nt 34078 and 35290 of {lambda} DNA) were incubated in a bath at 75 °C for 3 min and then TaqSSB protein was added. The final concentrations of DNAs and protein were 2–5 ng µl-1 and 1–5 ng µl-1, respectively. Controls with DNA or protein alone were incubated under the same conditions. Preparations for electron microscopy were made according to a previously described method (Szalewska-Palasz et al., 1998 ) and were analysed with a Philips CM 100 transmission electron microscope at 60 kV and a magnification of x39000.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Sequence analysis
The TthSSB and TaqSSB monomer proteins contain 264 and 266 amino acids, respectively. The homology between these proteins is very high: 82% identity and 90% similarity. They are the largest known prokaryotic SSB proteins (EcoSSB with N-terminal methionine is 178 amino acids long). A search in the Conserved Domain Database using RPS-BLAST (Madden et al., 1996 ) detected two putative ssDNA-binding sequences in the TthSSB and TaqSSB monomers: N-terminal (located in the region from amino acid 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively). The amino acid sequence alignment of thermostable SSB-like proteins from T. thermophilus HB-8 (this study, GenBank accession no. AF079160), T. thermophilus VK-1 (GenBank accession no. AF146075) and T. aquaticus YT-1 (this study, GenBank accession no. AF276705) with SSBs from mesophilic bacteria is presented in Fig. 1. To show the sequence identity of each ssDNA-binding domain of monomer of TthSSB and TaqSSB with other SSBs their sequences were divided into N- and C-terminal fragments. The sequence alignment of the N-terminal fragments of TthSSB and TaqSSB with the N-terminal 125 amino acids of EcoSSB (encoded by plasmid ColIb-P9) shows 32% and 33% identity, respectively. The longer C-terminal putative ssDNA-binding sequence (~140 amino acids) of the TthSSB and TaqSSB proteins possesses two regions of sequence similarity to EcoSSB (from ColIb-P9). The first region (amino acids 126–239), in analogy to the N-terminal domain, is also homologous to the N-terminal domain of EcoSSB (from ColIb-P9) (40 and 37% identity in TthSSB and TaqSSB, respectively). A second region of sequence identity in the C-terminal domain contains four conserved terminal amino acids: DIPF (Fig. 1).



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Fig. 1. Multiple amino acid sequence alignment of thermostable SSB-like proteins with SSBs from mesophilic bacteria. To show sequence similarity of each ssDNA-binding domain of the monomer of TthSSB and TaqSSB to other SSBs their sequences were divided into N- and C-terminal fragments. Alignment was performed by dividing amino acids into six similarity groups: group 1, V, L, I and M; group 2, W, F and Y; group 3, E and D; group 4, K and R; group 5, Q and D; group 6, S and T. Description of similarity: white fonts on black boxes, 100% identity; white fonts on grey boxes, similarity <80%; black fonts on grey boxes, similarity <60%. Asterisks indicate conserved amino acids in the EcoSSB sequence (E. coli) that is engaged in stacking interaction with bases of ssDNA (W40, W54, F60 and W88) and stabilization of the tetramer (H55). The arrows, bar and lines show ß-sheets, {alpha}-helix and loops, respectively, identified in the structure of EcoSSB (Matsumoto et al., 2000 ; Raghunathan et al., 1997 ). The assignment of secondary structures is marked according to the OB fold rule (Murzin, 1993 ). Abbreviations: Tth HB-8-N or -C: T. thermophilus strain HB-8, N- or C-terminal fragment; Tth VK1-N or -C: T. thermophilus strain VK-1, N- or C-terminal fragment; Taq YT1-N or -C: T. aquaticus strain YT-1, N- or C-terminal fragment; Drad: Deinococcus radiodurans strain R1; Paer: Pseudomonas aeruginosa strain PAO1; Hinf: Haemophilus influenzae; ColIb: Escherichia coli plasmid ColIb-P9; Bhld: Bacillus halodurans; Aqal: Aquifex aeolicus strain Rd KW20; Ecoli: Escherichia coli K-12.

 
DNA-binding properties
To determine the ability of thermostable SSB-like proteins to bind to ssDNA, we carried out agarose gel mobility assays with fluorescein-labelled ss dT-oligonucleotides 30, 60, 70, 76 nucleotides in length (Fig. 2). When (dT)30 and (dT)60 were incubated with increasing concentrations of SSB-like proteins, a single band of reduced mobility was observed. Most of the (dT)30 and (dT)60 was shifted after addition of one molar equivalent of TaqSSB or TthSSB (both calculated as 60 kDa homodimer), and the mobility of the shifted band remained constant at higher protein concentrations. A band of identical mobility was observed for (dT)70 and (dT)76 at low oligonucleotide concentrations, but a second band with a lower mobility was observed at high protein concentrations. These results suggest that TaqSSB and TthSSB proteins bind to (dT)30 and (dT)60 as a single protein molecule whereas two SSB molecules bind to (dT)70 and (dT)76. Thus, the ssDNA-binding site for TaqSSB and TthSSB protein is between 30 and 35 nucleotides long.



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Fig. 2. Binding of TthSSB to single-stranded oligonucleotides. A fixed quantity (20 pmol) of 5'-end fluorescein-labelled oligonucleotides (dT)30, (dT)60, (dT)70 or (dT)76 was incubated with 0, 5, 20, 50 and 200 pmol of TthSSB in 20 µl reaction mixtures for 5 min at 25 °C. Protein–DNA complexes were separated from free DNA by agarose gel electrophoresis.

 
An important biophysical property of SSB proteins is their ability to destabilize double-stranded DNA. Fig. 3 shows the destabilization of duplex DNA (44 bp) by both TaqSSB and TthSSB. Both proteins destabilize and lower the melting point temperature of duplex DNA in 0·1 M NaCl by 13 °C, similar to EcoSSB (Genschel et al., 1996 ).



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Fig. 3. Melting profiles of dsDNA and its complexes with TaqSSB and TthSSB. A 1 pmol sample of duplex DNA (44 bp) was incubated alone ({lozenge}) and with 2·5 pmol of TaqSSB (x) or TthSSB ({circ}) in standard buffer containing 0·1 M NaCl. Transition midpoints are at 75 °C (duplex DNA) and 62 °C (TaqSSB and TthSSB). Absorbance changes were measured at 260 nm.

 
To further explore the binding properties of TaqSSB and TthSSB proteins, we made use of fluorescence spectroscopy. We observed that the intrinsic fluorescence of thermostable SSB-like proteins, like that of EcoSSB protein, is quenched on DNA binding (Dabrowski & Kur, 1999 ). With an excitation wavelength of 295 nm, the emission spectrum of SSB-like proteins at 25 °C and 60 °C had a maximum at 348 nm, consistent with tryptophan fluorescence. On addition of a saturating quantity of ssDNA, the intrinsic fluorescence at 348 nm was quenched by 68% for TaqSSB (Fig. 4) and TthSSB (data not shown). Thus, eight of the twelve Trp residues of TaqSSB or TthSSB homodimer are directly engaged in ssDNA binding. The estimated size of the ssDNA binding site in the presence of 0·1 M NaCl for both thermostable SSB-like proteins is the same: 34±2 nt. For TaqSSB and TthSSB a binding-mode transition has been observed when changing the ionic environment from low salt (0·002 M NaCl) to high salt (0·3 M NaCl). A similar transition was observed for TaqSSB (Fig. 4) and TthSSB (data not shown), where the binding-site size in the presence of NaCl (>0·015 M) increases from 24±2 nt to 34±2 nt (the apparent EcoSSB binding-site size increases from 33 to 64 nt per tetramer: Lohman & Overman, 1985 ).



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Fig. 4. Inverse fluorescence titration of TaqSSB with (dT)75. A 0·4 µmol sample of SSB protein (homodimer) was titrated with (dT)75 at 0·1 M ({blacksquare}) and 0·002 M ({bullet}) NaCl in standard buffer. The binding-site size and fluorescence quenching for TaqSSB were calculated to be 34±2 nucleotides per homodimer and 68% at 0·1 M NaCl, and 24±2 nucleotides per homodimer and 59% at 0·002 M NaCl.

 
The kinetics of binding to poly(dT) can be observed by fluorescence changes in a stopped-flow experiment. Using a kinetic model for the binding of a multidentate ligand to a linear polymer described earlier (Urbanke & Schaper, 1990 ) the association-rate constants for the binding of protein homodimer to its binding site were determined to be (2·1±0·3)x108 M-1 s-1 for TaqSSB and (2·2±0·3)x108 M-1 s-1 for TthSSB. These values do not differ significantly from the values determined for EcoSSB (Urbanke & Schaper, 1990 ). We did not observe a difference in the kinetics of TaqSSB or TthSSB binding to (dT)60 or (dT)75 oligonucleotides.

Native molecular mass and thermostability
Analysis of the purified proteins by SDS-PAGE revealed single major bands with a molecular mass of 30 kDa (calculated from the amino acid sequences the molecular masses of TthSSB and TaqSSB are 29·87 and 30·03 kDa, respectively). Analysis of purified proteins by gel-permeation chromatography revealed single peaks with a molecular mass of about 60 kDa (60·2 and 60·5 for TthSSB and TaqSSB, respectively). The results show that TaqSSB and TthSSB are homodimers. Chemical cross-linking using glutaraldehyde and analytical ultracentrifugation studies at protein concentrations between 50 and 500 µg ml-1 confirmed the dimeric state of the proteins.

The half-lifes of ssDNA-binding activity of TaqSSB and TthSSB proteins at 80, 85, 90 and 95 °C were determined by the gel mobility shift assay of (dT)30 in 2% agarose gel (Fig. 5). The results obtained were surprising because the half-life of ssDNA-binding activity at 95 °C is only 30 s, while the half-life of Taq DNA polymerase activity (from T. aquaticus) at 95 °C is over 20 min (Korolev et al., 1995).



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Fig. 5. Half-lifes of ssDNA-binding activity of TaqSSB and TthSSB. A fixed quantity (20 pmol) of 5'-end fluorescein-labelled oligonucleotide (dT)30 was incubated with 20 pmol of TaqSSB ({square}) or TthSSB ({circ}) in 20 µl reaction mixtures in standard buffer (0·1 M NaCl) for 0, 5, 10, 15, 30 and 45 min at 80, 85, 90 and 95 °C. Protein–DNA complexes were separated from free DNA by 2% agarose gel electrophoresis, and 50% quantities of protein–(dT)30 complex were evaluated by densitometric analysis using BioDoc software (Biometra).

 
When analysed by differential scanning microcalorimetry (DSC) in 50 mM potassium phosphate buffer, pH 7·5, containing 0·1 M NaCl, the thermal unfolding of TaqSSB and TthSSB was found to be an irreversible process. TaqSSB showed slight higher thermostability, with a melting temperature (Tm) of 90·1 °C, whereas TthSSB has a Tm of 89·2 °C. This difference in Tm is also observed in the half-life of ssDNA binding assay (Fig. 5). Moreover, the results of DSC and the half-life of ssDNA binding assay suggest that the loss of binding activity of TaqSSB and TthSSB is connected with irreversible thermal unfolding of these proteins.

Electron microscopy
Electron microscopy analyses of TaqSSB–DNA complexes (Fig. 6) revealed that TaqSSB proteins bind with high affinity and thus completely coat ssDNA [poly (dT)700] (Fig. 6a). No such binding is observed to the dsDNA (Fig. 6c). It can be seen that the apparent ssDNA length becomes shortened; this is because TaqSSB can wrap ssDNA around itself.



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Fig. 6. Electron micrographic visualization of (a) TaqSSB protein binding to poly (dT)700, (b) TaqSSB protein alone, and (c) bacteriophage {lambda} dsDNA 1212 bp fragments in the presence of TaqSSB protein. Bars, 100 nm.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have reported here the identification and characterization of the SSB-like proteins of the thermophilic bacteria T. aquaticus and T. thermophilus. The results obtained revealed that the TthSSB and TaqSSB proteins belong to a new class of SSBs. We found that these proteins, in contrast to their known counterparts from mesophilic bacteria, archaea and eukarya, are homodimers. Sequence analysis showed that the monomers of TthSSB and TaqSSB contain two ssDNA-binding domains, each with the canonical OB fold. This analysis indicates that we may divide TthSSB and TaqSSB monomer into two putative domains (Fig. 1): a smaller N-terminal domain with a fragment responsible only for binding to ssDNA, and a larger C-terminal domain with a second ssDNA-binding sequence followed by a highly acidic fragment ~20 amino acids long. So, the functional dimer of TthSSB and TaqSSB proteins possesses four ssDNA-binding domains as in other prokaryotes and only two C-terminal regions with a putative role in interactions with other cellular proteins.

Sequence alignment shows a different similarity pattern in the N- and C-terminal ssDNA-binding domains of the TthSSB and TaqSSB monomers relative to critical residues involved in interaction with ssDNA in EcoSSB (Raghunathan et al., 2000 ). Each ssDNA-binding domain of TthSSB and TaqSSB possesses three Trp residues (as in E. coli). Only Trp-80 of EcoSSB is homologous to the corresponding Trp residue in the N-and C-terminal ssDNA-binding domains of the TthSSB and TaqSSB monomer. In place of the Trp-54 of EcoSSB, the N-terminal ssDNA-binding domain of TthSSB and TaqSSB possesses at the homologous position the aromatic Tyr preceded by Trp, and the C-terminal domain possesses the aromatic Phe residue. The Trp-40 of EcoSSB is homologous only to the Phe residue in the N-terminal domain of TaqSSB. The conserved Phe-60 of EcoSSB is homologous to the Trp residue in the C-terminal domains of TthSSB and TaqSSB. His-55, critical for the formation of the tetrameric form of SSB in E. coli and other prokaryotes (Williams et al., 1983 ), is homologous to the corresponding His residue present only in the N-terminal domains of the TaqSSB and TthSSB proteins. The N- and C-terminal ssDNA-binding domain of the monomer also differs in the distribution of the charged amino acids residues (Fig. 1). High sequence identity between TaqSSB, TthSSB and other known bacterial SSB proteins is observed for Gly and Pro residues. These residues are located in the structure of EcoSSB mainly in loops and turn regions. Conservation is observed also in the case of hydrophobic residues, which are responsible for stabilization of the protein core in EcoSSB (Matsumoto et al., 2000 ; Raghunathan et al., 1997 , 2000 ).

The C-terminal 24 amino acid domain of the TthSSB and TaqSSB proteins possesses the lowest sequence similarity to EcoSSB. This region in the crystal structure of EcoSSB protrudes from the surface of each subunit as an unstructured fragment due to the high content of Gly and Pro residues (Matsumoto et al., 2000 ). The function of this region in the interaction with other proteins is dependent on its acidic character, as shown by the construction of chimeric proteins (Curth et al., 1996 ; Handa et al., 2001 ). In TthSSB and TaqSSB proteins this region possesses conserved acidic character and a high content of Gly and Pro. A special feature observed in this region in the TthSSB and TaqSSB is a shortening of the spacer sequence, which in the SSB proteins of the mesophilic bacteria separates the ssDNA-binding domain from the acidic C-terminus (Fig. 1).

The SSBs from T. aquaticus and T. thermophilus do not possess any special features relative to the SSB of the mesophile E. coli. Although only the analysis of the structure of thermostable protein may explain the cause of the stability, some interesting information may be obtained from simple analysis of the sequence. Sequence comparisons of thermophilic and mesophilic proteins have shown some significant substitutions in thermophilic proteins such as Lys to Arg, Ser to Ala, Gly to Ala, Ser to Thr and Val to Ile (Ladenstein & Antranikian, 1998 ; Scandurra et al., 1998 ). The TthSSB and TaqSSB proteins have a content of charged (Asp, Glu, Lys and Arg) residues much higher than that of EcoSSB (Table 1). These residues in the thermophilic SSBs may be involved in stabilization of the interdomain surface by ionic networks. The higher content of positively charged residues may also suggest that the thermophilic SSB proteins interact much more strongly than EcoSSB with the backbone atoms of ssDNA. There is a significant increase of Arg residues and decrease of Lys residues relative to the EcoSSB (Table 1). Statistical analysis comparing the mean content of Arg shows 5·6% Arg in the amino acid compositions of thermophilic proteins, which is 1·24 percentage points higher than in mesophilic proteins (Vieille & Zeikus, 2001 ). In the thermophilic SSB proteins the percentage content of Arg is 7·6 percentage points higher than in EcoSSB. In many cases Arg residues stabilize proteins by hydrophobic interaction at the surface (van den Burg et al., 1994 ). As an ionic positively charged residue, Arg is better adapted to high temperatures than Lys. The {delta}-guanidino moiety provides more surface area for charged interaction than any other ionic residue. The pKa value of Arg is 12 and is approximately 1 unit above the pKa of Lys, so Arg maintains ion pairs and positive charge more easily at high temperatures than Lys. Thermostable SSB-like proteins possess a higher content of aliphatic hydrophobic residues than EcoSSB; there is a significant increase in the level of Leu (Table 1).


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Table 1. Percentage amino acid content of SSB proteins from bacteria

 
In many thermostable proteins, there is a preference for a decrease in the Gly content and an increase in the Pro content in positions of low structural importance for fold conservation (Korolev et al., 1995 ; Matthews et al., 1987 ). TthSSB and TaqSSB have a much lower content of Gly residues than EcoSSB (Table 1). Most of the Gly residues of the thermostable SSB proteins are homologous to conserved Gly in the EcoSSB (Fig. 1), which are located in sites responsible for correct loop formation.

Local unstructured regions of the protein are potential sites of denaturation (Hardy et al., 1993 ). In the structure of EcoSSB, a long C-terminal fragment protruding from the ssDNA-binding domain is such a potential site (Matsumoto et al., 2000 ). In comparison to EcoSSB, the thermostable SSBs possess a much shorter C-terminal fragment. It was shown for EcoSSB that truncation of the C-terminal fragment increases the strength of the interaction of the N-terminal domain with ssDNA (Curth et al., 1996 ). The thermostable SSB-like proteins possess only two acidic C-terminal fragments, as compared to four C-terminal fragments in the mesophilic SSB. This reduction may be an important adaptation of TaqSSB and TthSSB to extreme conditions.

In the thermostable SSBs one monomer protein possesses two ssDNA-binding domains that are reminiscent of two monomers of the mesophilic SSB. This conjunction in the thermostable SSB monomer may permit a reduction in the surface to volume ratio and more compactness in the hydrophobic core and in the interface between domains relative to the dimer in the mesophilic SSB (Ladenstein & Antranikian, 1998 ).


   ACKNOWLEDGEMENTS
 
The work was supported by the State Committee for Scientific Research Grant no. 4 PO5A09917 to J.K. We thank Dr W. Szybalski (University of Wisconsin, Madison, USA) for careful reading of and helpful comments concerning the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 19 March 2002; revised 31 May 2002; accepted 6 June 2002.