ATPase activity of UvrB protein form Thermus thermophilus HB8 and its interaction with DNA.

Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system. The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins. The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis. The T. thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity. The purified T. thermophilus UvrB protein was stable up to 80 degrees C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. coli UvrB protein alone has not been shown to exhibit detectable ATPase activity. The values of K(m) and k(cat) for the ATPase activity were 4.2 mM and 0.32 s-1 without DNA, and 4.0 mM and 0.46 s-1 with single-stranded DNA, respectively. This suggests that T. thermophilus UvrB protein could interact with single-stranded DNA in the absence of UvrA protein.

UvrB incises the 3Ј side of the DNA lesion and UvrC incises the 5Ј side (8). Finally, DNA helicase II, DNA polymerase I, and DNA ligase complete the repair (5).
In the above model, UvrA recognizes the damaged site in the DNA and guides UvrB to it. However, Hsu et al. (9) showed that UvrB can bind to DNA in the absence of UvrA. Although this finding indicates that UvrB plays a central role in ABC excinuclease activity, the details of the reaction mechanism are not clear. In order to understand the molecular mechanism of this enzyme's action in detail, enzymatic studies and physicochemical approaches, including x-ray crystallography and nuclear magnetic resonance, are required.
Thermus thermophilus HB8 is an aerobic, rod-shaped, nonsporulating, Gram-negative eubacterium, which can grow at temperatures over 75°C (10). Although hyper-thermophilic bacteria can grow at temperatures over 100°C (such as Pyrococcus fuliosus, 11), T. thermophilus is the most thermophilic bacterium whose gene manipulating system has been established among thermophilic bacteria. Its proteins are heatstable and easily crystallized (12)(13)(14), so they are suitable for detailed physicochemical research.
In this study, we cloned and sequenced the uvrB gene from T. thermophilus. The gene was overexpressed in E. coli, and its product was purified to homogeneity. ATPase activity in the absence and presence of DNA and stability of T. thermophilus UvrB protein were also reported. To our knowledge, this is the first demonstration of ATPase activity of this UvrB protein itself, whereas its E. coli counterpart has not been shown to exhibit detectable ATPase activity. We also found that the UvrB protein could interact with single-stranded DNA (ssDNA) 1 in the absence of UvrA.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D49912.
Cloning and Sequencing of the T. thermophilus uvrB Gene-The amino acid sequences of UvrB proteins from E. coli (19) and Micrococcus luteus (20) were aligned, and two highly conserved regions were chosen as the bases for synthesizing oligonucleotide primers for the polymerase chain reaction (PCR) (21). The left and right mixed primers were 5Ј-TACTACGACTACTA(C/T)CA(G/A)CC-3Ј (20 mer) and 5Ј-ATCTG(G/C)GGGAT(G/C)GT(G/C)AC(G/A)TG-3Ј (20 mer), respectively. T. thermophilus genomic DNA (0.2 g) was added to the PCR mixture and then denatured at 98°C for 10 min without DNA polymerase. Following the addition of Taq DNA polymerase, the PCR was carried out for 30 cycles of 1 min at 98°C, 1 min at 50°C, and 2 min at 72°C with a 3-min ramp time. The nucleotide sequence of the amplified DNA fragment, which was purified by agarose gel electrophoresis, was determined to confirm that the translated amino acid sequence was similar to that of the UvrB protein. The amplified DNA fragment ( Fig.  1, b-1) was cloned into pUC118 for use as a probe. T. thermophilus genomic DNA was digested with several restriction endonucleases and then subjected to Southern hybridization. The DNA fragments that hybridized with the probe were purified and ligated with pUC119 to construct a mini-gene bank. The E. coli cells transformed with the plasmids were screened by colony hybridization and a 6.5-kilobase pair positive DNA fragment ( Fig. 1, pNB1) was obtained. Further screening was carried out using a BglII-BamHI fragment of pNB1 as a probe ( Fig.  1, b-2) to clone the C-terminal region of the gene and a DNA fragment, pNB2 ( Fig. 1), was obtained. A DNA fragment containing the entire T. thermophilus uvrB gene was then constructed by ligation of parts of pNB1 and pNB2 (Fig. 1, pNB3). The nucleotide sequence of pNB3 was determined on both strands by the dideoxy method (Applied Biosystems, Taq cycle sequencing system) using an automated DNA sequencer (Applied Biosystems, model 373A). To prevent the pausing of sequencing reaction, secondary structure of T. thermophilus DNA, which has a high GϩC content, was destroyed by heat denaturation (98°C, 10 min) prior to the sequencing reaction.
Phylogenetic Calculation-Molecular evolutionary calculations for construction of a phylogenetic tree were performed with the aid of the ODEN program package written by Y. Ina of the National Institute of Genetics (Mishima, Japan).
Overexpression of the T. thermophilus uvrB Gene in E. coli-A pair of DNA primers, 5Ј-CCCTATACTGGGTGCATATGACCTTCCGCTAC-3Ј and 5Ј-GGGCGAGGACCAAAGCGGGCCTTC-3Ј were synthesized to create a NdeI restriction endonuclease site for the first ATG codon of the T. thermophilus uvrB gene. The underlining of the former primer indicates the NdeI site and the latter contained a unique restriction endonuclease site, NcoI, 200 base pairs downstream from the first ATG. A DNA fragment of about 200 base pairs was amplified by PCR using pNB3 as a template, under the conditions described above. The amplified fragment was sequenced to confirm that no mutation other than at the NdeI site was present. Then, the DNA fragments of NdeI-NcoI of the PCR product, NcoI-blunt ended BlnI of pNB3 and NdeI-blunt ended BamHI of pFT3a were ligated. The resulting plasmid was named pYB1 and used to study expression in E. coli cells. The T. thermophilus uvrB gene was not overexpressed in E. coli when it was inserted under control of the T7 promoter (data not shown). The co-existence of the pLysE plasmid, which represses the basal expression of the gene, was required for overproduction of the protein. The E. coli strain BL21(DE3) pLysE harboring plasmid pYB1 was cultivated at 37°C in LB medium containing 50 g/ml ampicillin and 20 g/ml chloramphenicol. When the cells had grown to 4 ϫ 10 8 cells/ml, IPTG was added to produce a concentration of 50 g/ml. After an additional 3 h of incubation, the cells were harvested and stored at Ϫ80°C.
Purification of T. thermophilus UvrB Protein-Frozen cells (20 g) were thawed and suspended in buffer I (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 8 mM ␤-mercaptoethanol, 25% (w/v) sucrose). They were sonicated on ice for 4 min with an ultrasonic disrupter (Tomy, UD201) at maximum output with a standard tip. Then Brij-58 was added to a final concentration of 0.2% (w/v) and held on ice for 30 min. The cell extract was incubated at 70°C for 20 min and then centrifuged (48,000 ϫ g) for 20 min at 4°C. At this step, most of the E. coli proteins could be excluded. The supernatant was dialyzed against buffer II (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM ␤-mercaptoethanol, 10% (v/v) glycerol) containing 100 mM KCl and then loaded onto a column of DEAEcellulose (bed volume 200 ml) equilibrated with this buffer. The column was washed with 250 ml buffer II containing 150 mM KCl and then eluted with a 1,000 ml gradient of 150 -500 mM KCl in buffer II. The eluate was monitored by absorption at 280 nm with a spectrophotometer (Hitachi, model 323), and the fractions around the peaks were analyzed by SDS-polyacrylamide gel electrophoresis. The fractions containing UvrB protein, which was eluted at 300 mM KCl, were pooled and dialyzed against buffer II containing 10% (w/v) ammonium sulfate. The sample was loaded onto a column of Phenyl-Toyopearl 650 M (bed volume 80 ml) equilibrated with this buffer; the column was eluted with a 500-ml gradient of 10 -0% (w/v) ammonium sulfate in buffer II, and then washed with an additional 100 ml of buffer II. The fractions were analyzed as described above, and UvrB protein was eluted at 0% ammonium sulfate. The fractions containing the protein were pooled and dialyzed against buffer III (1 mM EDTA, 10 mM ␤-mercaptoethanol, 10% (v/v) glycerol) containing 10 mM potassium phosphate (pH 6.8), and then loaded onto a column of hydroxylapatite (bed volume 80 ml) equilibrated with this buffer. The column was eluted with a 400 ml gradient of 10 -200 mM potassium phosphate (pH 6.8) in buffer III. The fractions were analyzed as described above, and UvrB protein was eluted at 120 mM potassium phosphate. These fractions were pooled and dialyzed against buffer II containing 100 mM KCl, and then stored at 4°C. The amount of purified UvrB protein obtained was 40 mg, and the yield was 2 mg of protein/1 g of E. coli cells (wet weight).
The purity of the final fraction of T. thermophilus UvrB protein was over 99%, based on the densitometry of SDS-polyacrylamide gel stained with Coomassie Brilliant Blue (CBB) (Fig. 4B, lane 5) or silver-staining, N-terminal amino acid sequencing analysis and the size exclusion chromatography (Fig. 8). The final T. thermophilus UvrB fraction was extremely pure, as shown by the observation of no contamination bands on the gel by silver-staining, which is 100 times more sensitive than ordinary CBB staining (22).
Amino Acid Sequencing-To determine the N-terminal amino acid sequence of T. thermophilus UvrB protein, the protein separated on the SDS-polyacrylamide gel was electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad) at 1 mA/cm 2 for 4 h. The band of the protein visualized using CBB was cut out and analyzed by a protein sequencer (Applied Biosystems, model 473A). The C-terminal sequence of the purified T. thermophilus UvrB protein was analyzed by digesting the protein with carboxypeptidase A or B (23) and detection of the released amino acids using an amino acid analyzer (Irica, model A-5500).
Determination of Protein Concentration-T. thermophilus UvrB protein contains 3 tryptophan and 29 tyrosine residues (see Fig. 2). The molar extinction coefficient was calculated to be 60,000 M Ϫ1 ⅐cm Ϫ1 at an absorption maximum around 277 nm using the procedure described previously (24).
Circular Dichroic (CD) Measurement-CD spectra were obtained using 20 mM Tris-HCl (pH 7.5) and 2 M T. thermophilus UvrB protein in a 0.1-cm cell (far-UV region between 200 and 250 nm) or 20 M protein in a 0.5-cm cell (near-UV region between 250 and 320 nm), at 25°C. The residue molar ellipticity, [], was defined as 100⅐ obs ⅐(lc) Ϫ1 , where obs is the observed molar ellipticity, l is the length of the light path in cm, and c is the residue molar concentration of T. thermophilus UvrB protein. CD measurements were performed using a Jasco spectropolarimeter, model J-500A, equipped with an interface and a computer.
ATPase Assay-Hydrolysis of ATP by T. thermophilus UvrB protein was measured at 65°C by thin-layer chromatography (25). Each reaction mixture (total volume 10 l) comprised 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM dithiothreitol, 5 M T. thermophilus UvrB protein, 200 M poly(dC), which was used as ssDNA, and [␣-32 P]ATP (concentration as specified, 5.55 MBq/ml) as indicated concentration. One drop of mineral oil was floated on the top of the reaction mixture to prevent evaporation. Following a 5-min incubation, the reaction was stopped by adding 2.5 l of 10% (w/v) SDS. After dissolving the oil with chloroform, 0.3-l aliquots were spotted onto polyethyleneimine cellulose sheets and eluted with 0.75 M potassium phosphate (pH 3.4). The sheets were placed in contact with imaging plates for 20 min, and then the radioactive counts due to ATP and ADP were determined using a BAS2000 image analyzer (Fuji photo film).

RESULTS AND DISCUSSION
Cloning, Sequencing, and Primary Structure of the T. thermophilus uvrB Gene-The amino acid sequences of previously determined UvrB proteins were aligned, and highly conserved regions of the proteins were chosen as bases for primer design. Using the synthesized primers, part of the T. thermophilus uvrB gene was amplified by the PCR. Using this DNA fragment (Fig. 1, b-1) as a probe, five positive colonies were screened from approximately 1,000 colonies of the mini-gene bank as described under "Materials and Methods." Plasmid DNA was prepared from each positive colony, all of which contained the same part of the uvrB gene (Fig. 1, pNB1). To clone the 3Ј region of the gene, we screened the other mini-gene bank using probe b-2 as described above, and obtained the DNA fragment pNB2 (Fig. 1). A plasmid, pNB3, which covered the entire T. thermophilus uvrB gene, was constructed from pNB1 and pNB2 (Fig. 1).
The nucleotide sequence of the T. thermophilus uvrB gene was determined, as described under "Materials and Methods," and its GϩC content was 68%, which agrees well with that of  a, b, and t, respectively. The ␣-helical region, residues 251-295 in T. thermophilus, was underlined (see text for details).
T. thermophilus HB8 genomic DNA (69%) (10). An open reading frame consisting of 1,995 nucleotides, which encoded a 665-amino acid protein with a calculated molecular mass of about 76.2 kDa was found. As shown in Fig. 2, the amino acid sequence of T. thermophilus UvrB protein is similar to that of other UvrB proteins. Comparison of the T. thermophilus UvrB protein with those of E. coli (19), M. luteus (20), Streptococcus pneumoniae (26), and Neisseria gonorrhoeae (27) revealed identities of 54, 52, 55, and 52% and similarities of 68, 66, 69, and 67%, respectively. The finding of the uvrB gene in T. thermophilus suggests that a UvrABC-like nucleotide excision repair system is present in this extremely thermophilic bacterium. The cloning of a UvrA homologue from T. thermophilus 2 leads support to this possibility.
Similarities between DNA/RNA Helicases and UvrB Protein-As described above, T. thermophilus UvrB protein showed high amino acid sequence similarities with its homologues. Walker's A-type nucleotide binding motif (28), GXXXXGK(T/S), and the helicase core sequence (29) DE(S/ A)(H/D) were also conserved (Fig. 2). DNA helicase activity has been detected by a E. coli UvrA-UvrB complex (30,31), and the helicase activity of the complex was suggested to be attributable to the UvrB protein since the complex formed from the mutant UvrB protein showed no such activity (32). DNA helicase activity may be a general requirement for DNA excision repair, because some proteins that are components of DNA repair complexes possess such activity.
The amino acid sequences of UvrB proteins and other DNA helicases were aligned and compared. Using the evolutionary distance values calculated by the method of Zuckerkandl and Pauling (33), a phylogenetic tree for the proteins was constructed by the neighbor-joining method of Saitou and Nei (34), as shown in Fig. 3. The tree suggests that these proteins can be classified into three groups. The first group comprises the UvrB proteins from different prokaryotic species. The second group consists of yeast Rad3 (35) and human XP-D (36, 37) proteins, which are components of the DNA excision repair system in eukaryotes. And the third group comprises E. coli SrmB (38), yeast p68 (39), murine PL10 (40), and human eIF-4AI (41) proteins, which are not related to DNA repair. UvrB, Rad3, and XP-D proteins are all components of DNA repair complexes, which are, however, located on different branches of the pedigree. The repair DNA helicases may have separated at an early evolutionary stage and then developed individually into the prokaryotic and eukaryotic DNA helicases in each repair complex.
Overproduction and Purification of T. thermophilus UvrB Protein-To analyze the biochemical properties of T. thermophilus UvrB protein, it was overproduced in E. coli and purified. The original clone of the T. thermophilus uvrB gene from pNB3 was subcloned into the expression vector pET3a, as described under "Materials and Methods." Only when a pLysE plasmid was co-transformed was an IPTG-induced band ob- served at a position of about 76 kDa, which was in agreement with the molecular mass calculated from the amino acid sequence of T. thermophilus UvrB protein (Fig. 4A).
Details of the purification of T. thermophilus UvrB protein are described under "Materials and Methods." Heat treatment prior to column chromatography was effective for removing most of the endogenous E. coli proteins. After heat treatment at 70°C for 20 min, the protein was purified to homogeneity using DEAE-cellulose, Phenyl-Toyopearl, and hydroxylapatite chromatography (Fig. 4B). T. thermophilus UvrB protein eluted at 0% (w/v) ammonium sulfate during Phenyl-Toyopearl chromatography, suggesting that this protein is highly hydrophobic.
The N-terminal amino acid sequence of the purified T. thermophilus UvrB protein overproduced in E. coli was found to be F-R-Y-R-G-P-S-P-K-G-, and the gene product was truncated, lacking two residues from the N terminus in comparison with the expected sequence from the nucleotide sequence, although the remaining sequence was identical to that expected (Fig. 2). This N-terminal truncation of the protein did not occur during protein purification since the protein in the total cell lysate before purification blotted onto the polyvinylidene difluoride membrane already lacked the two N-terminal amino acid residues. It is unknown whether such processing of the UvrB protein occurs in the T. thermophilus cell. The amino acid composition of the C-terminal region of the protein was determined and was the same as that predicted from the nucleotide sequence. As described below, the purified protein maintained its secondary structure, thermal resistance, and ATPase activity; therefore, it retains its native characteristics.
Secondary Structure-We attempted to characterize the purified T. thermophilus UvrB protein using various physicochemical approaches. First, we examined the far-UV CD spectrum to obtain information on the polypeptide backbone conformation. The far-UV CD spectrum of recombinant T. thermophilus UvrB protein had negative double maxima at around 210 and 220 nm (Fig. 5), which are characteristic of an ␣-helical structure. The helical content of T. thermophilus UvrB was estimated to be about 53% using the method of Chen et al. (42).
Summers et al. (43) have found that for proteins greater than 40% homology, at least 80% of the side-chain orientations are identical. Since the homologies among the five UvrB proteins in Fig. 2 were greater than 65%, these proteins are expected to have almost identical conformations. The common secondary structures among the five UvrB proteins including T. thermophilus, E. coli, M. luteus, S. pneumoniae, and N. gonorrhoeae were predicted by the method of Chou and Fasman (44) (Fig. 2). The ␣-helical and ␤-sheet contents of these UvrB proteins were estimated to be 54 and 12%, respectively. This predicted ␣-helical content is well agreed with the above value estimated from the CD measurement. It should be noted that although the region between residues 251 and 295 in T. thermophilus has low homology among UvrB proteins, this region was predicted to have a high ␣-helical propensity. The ␣-helical conformation of this region will be essential for functional UvrB protein.
Stability of T. thermophilus UvrB Protein-We used the residue molar ellipticity at 222 nm ([] 222 ) to determine the thermal stability of T. thermophilus UvrB protein. As shown in Fig.  6, the protein was stable from 5 to 80°C. Above 80°C, it was denatured and aggregated and this thermal denaturation was irreversible. The ATPase activity of T. thermophilus UvrB protein was retained from 4°C to 80°C (data not shown). In an attempt to elucidate the relationship between thermostability and amino acid residue replacement, we compared the thermophilic and mesophilic enzymes. The sequence homology of T. thermophilus and E. coli UvrB proteins was 68%, and some interesting amino acid substitutions were evident. There were less Met and Asn residues in T. thermophilus than E. coli UvrB protein. These residues are known to be chemically unstable at high temperature. The number of Pro residues had increased from 33 in E. coli UvrB protein to 39 in T. thermophilus. Generally, Pro residues are considered to decrease the entropy of the denatured state of the protein or to increase the conformational energy in the native state, thereby increasing protein stability (45). The number of Arg residues increased from 52 in E. coli to 76 in T. thermophilus. These changes were also observed in other thermostable proteins (16, 46 -48).
Next, the effect of pH on T. thermophilus UvrB protein stability was studied on the basis of [] 222 . It was stable between pH 6 and 11, whereas it aggregated between pH 4 and 6 and its ellipticity could not be measured (Fig. 7). The [] 222 at pH 4 was similar to that at pH 6, and the protein denatured gradually at lower pH values. Many other proteins from Thermus species are also stable at highly alkaline pH values (49 -52). 3 Although this stability at high pH values is a common feature of thermophilic proteins, the reason for it is not clear.
Protein-Protein Interaction of UvrB-According to the results of SDS-polyacrylamide gel electrophoresis, the molecular 3 R. Kato  mass of T. thermophilus UvrB protein was about 76 kDa (Fig.  4), which was consistent with the value calculated from the amino acid sequence. We performed size-exclusion chromatography to determine the molecular mass of the protein in its native (not denatured) state and whether it is a monomer or multimer. As shown in Fig. 8, estimated molecular mass of T. thermophilus UvrB protein was between about 70 and 80 kDa, which suggests that the protein exists as a monomer at neutral pH. Although T. thermophilus UvrB protein was a monomer in the absence of SDS at neutral pH, it aggregated between pH 4 and 6. The isoelectric point of T. thermophilus UvrB protein, calculated from its amino acid sequence, is 5.5; therefore, its net charge may approach zero at pH 4 -6. As electrostatic repulsion is weakened when the net charge of the molecule is lost, protein aggregation may occur around its isoelectric point.
The Phenyl-Toyopearl column chromatographic behavior of T. thermophilus UvrB protein during purification indicated that the protein is hydrophobic and that some hydrophobic residues will be exposed to the solvent. The hydrophobic surface of T. thermophilus UvrB protein molecule may lead to self-aggregation around its isoelectric point and may contribute to the protein-protein interaction between UvrB and UvrA or UvrC.
ATPase Activity-Many proteins that have Walker's A-type motif show ATPase activity, and this motif was also conserved in UvrB proteins (Fig. 2). However, E. coli UvrB protein has been believed that it has no ATPase activity in the absence of UvrA protein (53). To clarify whether T. thermophilus UvrB protein has ATPase activity, we used thin-layer chromatography to separate the reaction products obtained after incubating the purified T. thermophilus UvrB protein with [␣-32 P]ATP. As shown in Fig. 9, T. thermophilus UvrB hydrolyzed ATP at 65°C, and this activity was stimulated by ssDNA, poly(dC). This ssDNA-stimulated ATPase activity differed from that of E. coli UvrB protein, the ATPase activity of which is cryptic in the absence of UvrA protein.
To determine the K m and k cat of T. thermophilus UvrB protein, ATPase activity was measured in the presence of various concentrations of ATP (Fig. 9B). The calculated values are summarized in Table I. The k cat value in the presence of ssDNA was about 1.5 times greater than its absence, whereas K m was almost the same under both conditions. These findings suggest that the binding of ssDNA affects the catalytic groups of UvrB protein but not does affect the nucleotide binding affinity of the protein. Although the K m and k cat values of E. coli UvrB protein has not been determined because it alone has no detectable ATPase activity, the values of E. coli UvrA-UvrB complex were determined (53). The k cat value of the complex is increased by addition of ssDNA (from 0.32 s Ϫ1 to 10.2 s Ϫ1 ), while the K m is changed from 0.21 mM to 1.5 mM (Table I).
It was reported that, during isolation, E. coli UvrB protein  was susceptible to protease, yielding a 70-kDa protein, UvrB*, which was probably cleaved by Ada protease near the C terminus (5,53). This UvrB* protein showed ATPase activity in the absence of UvrA, and this activity was stimulated by ssDNA (53). Although T. thermophilus UvrB and E. coli UvrB* both showed ssDNA-stimulated ATPase activity, the former has no potential cleavage site for Ada protease and the C terminus of the purified protein is not processed. Therefore, the ATPase activity of T. thermophilus UvrB protein is not artificial, but a natural property. The activity of T. thermophilus UvrB protein became obvious at a higher temperature, 65°C. Although E. coli UvrB protein had no ATPase activity in the absence of UvrA at 37°C (53), its ATPase activity may be measurable if E. coli UvrB protein was also stable up to 65°C. A recent study showed that E. coli UvrB protein, at high concentration, could bind to damaged DNA in the absence of UvrA (9). These observations suggest strongly that UvrB itself possesses its own activity in the absence of UvrA.
Since the ATPase activity of T. thermophilus UvrB protein was stimulated by ssDNA, this protein may interact with ssDNA. In the accepted model of nucleotide excision repair, UvrB protein interacts with damaged DNA in the UvrA 2 B-DNA complex, called an open complex, in which the DNA is unwound. Therefore, the interaction of T. thermophilus UvrB protein with ssDNA may correspond to its binding to the unwound DNA in the open complex, and therefore, UvrB protein may interact directly with ssDNA in the open complex without the assistance of UvrA protein during nucleotide excision repair.