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
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ABSTRACT |
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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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INTRODUCTION |
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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.
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METHODS |
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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 LuriaBertani 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 proteinprotein 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 120 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 (Dbrowski & 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 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 25 ng µl-1 and 15 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-Pa
asz et al., 1998
) and were analysed with a Philips CM 100 transmission electron microscope at 60 kV and a magnification of x39000.
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RESULTS |
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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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Electron microscopy
Electron microscopy analyses of TaqSSBDNA 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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DISCUSSION |
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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
-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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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 ).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Atrazhev, A., Zhang, S. & Grosse, F. (1992). Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-primase complex. Eur J Biochem 210, 855-865.[Abstract]
Augustyns, K., van Aerschot, A., van Shepdael, A., Urbanke, C. & Herdevijn, P. (1991). Influence of the incorporation of (S)-9-(3, 4-dihydroxybutyl)adenine on the enzymatic stability and base-pairing properties of oligodeoxynucleotides. Nucleic Acids Res 19, 2587-2593.[Abstract]
Bochkarev, A., Pfuetzner, R. A., Edwards, A. M. & Frappier, L. (1997). Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature 385, 176-181.[Medline]
Bochkarev, A., Bochkareva, E., Frappier, L. & Edwards, A. M. (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.
Bochkareva, E., Korolev, S. & Bochkarev, A. (2000). The role for zinc in replication protein A. J Biol Chem 275, 27332-27338.
Brill, S. J. & Stillman, B. (1991). Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev 5, 1589-1600.[Abstract]
Carlini, L., Curth, U., Kindler, B., Urbanke, C. & Porter, R. D. (1998). Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli. FEBS Lett 430, 197-200.[Medline]
Curth, U., Urbanke, C., Greipel, J., Gerberding, H., Tiranti, V. & Zeviani, M. (1994). Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties. Eur J Biochem 221, 435-443.[Abstract]
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.
Dbrowski, S. & Kur, J. (1999). Cloning and overexpression and purification of his-tagged SSB from E. coli and application in PCR. Protein Expr Purif 16, 96-102.[Medline]
de Vries, J. & Wackernagel, W. (1993). Cloning and sequencing of the Serratia marcescens gene encoding a single-stranded DNA-binding protein (SSB) and its promoter region. Gene 127, 39-45.[Medline]
de Vries, J. & Wackernagel, W. (1994). Cloning and sequencing of the Proteus mirabilis gene for a single-stranded DNA-binding protein (SSB) and complementation of Escherichia coli ssb point and deletion mutations. Microbiology 140, 889-895.[Abstract]
Genschel, J., Litz, L., Thole, H., Roemling, U. & Urbanke, C. (1996). Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO. Gene 182, 137-143.[Medline]
Gomes, X. V. & Wold, M. S. (1995). Structural analysis of human replication protein A. Mapping functional domains of the 70-kDa subunit. J Biol Chem 270, 4534-4543.
Gomes, X. V. & Wold, M. S. (1996). Functional domains of the 70-kilodalton subunit of human replication protein A. Biochemistry 35, 10558-10568.[Medline]
Greipel, J., Urbanke, C. & Maass, G. (1989). The single-stranded DNA binding protein of Escherichia coli. Physicochemical properties and biological functions. In ProteinNucleic Acid Interaction , pp. 61-86. Edited by W. Saenger & U. Heinemann. London: Macmillan.
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.
Hardy, F., Vriend, G., Veltman, O. R., van der Vinne, B., Venema, G. & Eijsink, V. G. H. (1993). Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett 317, 89-92.[Medline]
Kelly, T. J., Simancek, P. & Brush, G. S. (1998). Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii. Proc Natl Acad Sci USA 95, 14634-14639.
Kelman, Z., Pietrokovski, S. & Hurwitz, J. (1999). Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum H. J Biol Chem 274, 28751-28761.
Komori, K. & Ishino, Y. (2001). Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination. J Biol Chem 276, 25654-25660.
Korolev, S., Murad, N., Barnes, W. M., DiCera, E. & Waksman, G. (1995). Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2·5- resolution: structural basis for thermostability. Proc Natl Acad Sci USA 84, 9264-9268.
Kur, J., Hasan, N. & Szybalski, W. (1989). Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late pR' promoter and its mutants. Gene 81, 1-15.[Medline]
Ladenstein, R. & Antranikian, G. (1998). Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water. Adv Biochem Eng Biotechnol 61, 37-85.[Medline]
Lin, Y.-L., Shivji, K. K. M., Chen, C., Kolodner, R., Wood, R. R. & Dutta, A. (1998). The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair. J Biol Chem 273, 1453-1461.
Lohman, T. M. & Overman, L. B. (1985). Two binding modes in Escherichia coli single strand binding proteinsingle stranded DNA complexes. Modulation by NaCl concentration. J Biol Chem 260, 3594-3603.[Abstract]
Madden, T. L., Tatusov, R. L. & Zhang, J. (1996). Applications of network BLAST server. Methods Enzymol 266, 131-141.[Medline]
Matsumoto, T., Morimoto, Y., Shibata, N., Kinebuchi, T., Shimamoto, N., Tasukihara, T. & Yasuoka, N. (2000). Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-ray structure analysis. J Biochem 127, 329-335.[Abstract]
Matthews, B., Nicholson, H. & Becklet, W. J. (1987). Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 84, 6663-6667.[Abstract]
Meyer, R. R. & Laine, P. S. (1990). The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev 54, 342-380.
Moore, S. P., Erdile, T., Kelly, T. & Fishel, R. (1991). The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A. Proc Natl Acad Sci USA 88, 9067-9071.[Abstract]
Murzin, A. G. (1993). OB (oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12, 861-867.[Abstract]
Pfuetzner, R. A., Bochkarev, A., Frappier, L. & Edwards, A. M. (1997). Replication protein A. Characterization and crystallization of the DNA binding domain. J Biol Chem 272, 430-434.
Purnapatre, K. & Varshney, U. (1999). Cloning, over-expression and biochemical characterization of the single-stranded DNA binding protein from Mycobacterium tuberculosis. Eur J Biochem 264, 591-598.
Raghunathan, S., Ricard, C. S., Lohman, T. M. & Waksman, G. (1997). Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2. 9- resolution. Proc Natl Acad Sci USA 94, 6652-6657.
Raghunathan, S., Kozlov, A. G., Lohman, T. M. & Waksman, G. (2000). Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nat Struct Biol 7, 648-652.[Medline]
Rudolf, R., Böhm, G., Lilie, H. & Jaenicke, R. (1996). Folding proteins. In Protein Function: a Practical Approach. Edited by T. E. Creighton. Oxford: IRL Press.
Scandurra, R., Consalvi, V., Chiaraluce, R., Politi, L. & Engel, P. (1998). Protein thermostability in extremophiles. Biochimie 80, 933-941.[Medline]
Siegel, L. M. & Monty, K. J. (1966). Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta 112, 346-362.[Medline]
Smith, D. R., Doucette-Stamm, L. A., Deloughery, C. & 34 other authors (1997). Complete genome sequence of Methanobacterium thermoautotrophicum H: functional analysis and comparative genomics. J Bacteriol 179, 71357155.[Abstract]
Szalewska-Paasz, A., Weigel, C., Speck, C. & 7 other authors (1998). Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA. Mol Gen Genet 259, 678688.
Urbanke, C. & Schaper, A. (1990). Kinetics of binding of single-stranded DNA binding protein from Escherichia coli to single-stranded nuclei acids. Biochemistry 29, 1744-1749.[Medline]
van den Burg, B., Dijkstra, B. W., Vriend, G., van der Vinne, B., Venema, G. & Eijsink, V. G. H. (1994). Protein stabilization by hydrophobic interactions at the surface. Eur J Biochem 220, 981-985.[Abstract]
Vieille, C. & Zeikus, G. J. (2001). Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65, 1-43.
Webster, G., Genschel, J., Curth, U., Urbanke, C., Kang, C.-H. & 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.[Medline]
Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A. & Chase, J. W. (1983). Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. Evidence for a functionally homologous domain in both the Escherichia coli and T4 DNA binding proteins. J Biol Chem 258, 3346-3355.
Yang, C., Curth, U., Urbanke, C. & Kang, C.-H. (1997). Crystal structure of human mitochondrial single-stranded DNA binding protein at 2. 4 resolution. Nat Struct Biol 4, 153-157.[Medline]
Received 19 March 2002;
revised 31 May 2002;
accepted 6 June 2002.