(Received for publication, September 18, 1995; and in revised form, January 16, 1996)
From the
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 °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 and k
for the
ATPase activity were 4.2 mM and 0.32 s
without DNA, and 4.0 mM and 0.46 s
with single-stranded DNA, respectively. This suggests that T.
thermophilus UvrB protein could interact with single-stranded DNA
in the absence of UvrA protein.
All living organisms have DNA repair systems to counteract the
many forms of DNA damage due to sunlight, chemical agents, or ionizing
radiation(1) . If such damage is not repaired, mutagenesis or
even cell death may occur. The DNA repair systems involve in situ repair (photoreactivation), base excision repair, nucleotide
excision repair, mismatch repair, and recombinational
repair(1) . Of these, the nucleotide excision repair system can
deal with a wide range of DNA lesions. In Escherichia coli of
typical prokaryote, ABC excinuclease, which is encoded by the uvrA, B, and C genes, plays a major role in
this system(2, 3, 4, 5) . The
molecular mechanism of the nucleotide excision repair system, studied
mainly in E. coli, is as follows. First, UvrA, a damage
recognition protein, makes an UvrAB complex with UvrB (6) and then this complex binds to the site of the DNA lesion,
forming a UvrA
B-DNA complex(6, 7) , in
which, the DNA is unwound, creating a so-called ``open
complex.'' Next, UvrA dissociates from the complex and only UvrB
is bound to the DNA. Then, UvrC becomes associated with the complex,
forming an UvrBC-DNA complex(6) . At this point, UvrB incises
the 3` side of the DNA lesion and UvrC incises the 5` 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) ()in the absence
of UvrA.
Figure 1: Restriction endonuclease maps of cloned and subcloned DNA fragments. The thin lines indicate the DNA fragments cloned, the open arrow indicates the T. thermophilus uvrB open reading frame, and the bold lines b-1 and b-2 indicate the probes used for cloning (see text for details).
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) .
Figure 4:
Overproduction and purification of T.
thermophilus UvrB protein were analyzed by SDS-polyacrylamide gel
electrophoresis CBB staining(54) . Molecular mass markers
(Bio-Rad) are indicated to the left of each panel (E. coli -galactosidase, 116 kDa; rabbit phosphorylase b, 97
kDa; bovine serum albumin, 66 kDa; hen egg white ovalbumin, 45 kDa). A, analysis of overproduction of T. thermophilus UvrB
protein in E. coli. Lane 1, before induction by IPTG; lanes 2 and 3, 1.5 and 3 h after induction,
respectively. The arrow indicates T. thermophilus UvrB protein. The resolving gel contained 12% (w/v) of acrylamide. B, purification steps of T. thermophilus UvrB
protein. Lane 1, total cell extract; lane 2,
supernatant after heat treatment; lanes 3-5,
DEAE-cellulose, Phenyl-Toyopearl, and hydroxylapatite chromatography
fractions. The resolving gel contained 7.5% (w/v) of acrylamide. About
2-10 µg of protein was loaded into each lane of the
gel.
Figure 8:
Size-exclusion chromatography of T.
thermophilus UvrB protein. Measurements were performed at room
temperature using a buffer solution comprising 100 mM KCl and
10 mM Tris-HCl (pH 7.0). A column used was Pharmacia Superdex
200HR (1 cm 30 cm). 8 µl of 2.6 mg/ml UvrB protein was
injected to the column and analyzed by absorbance at 220 nm with a flow
rate of 0.5 ml/min. The molecular size markers (Sigma) are: BD, blue dextran; IgG, human immunoglobulin G (150
kDa); BSA, bovine serum albumin (64 kDa); OA, chicken
ovalbumin (44 kDa); CA, bovine carbonic anhydrase (29
kDa).
Figure 2:
Amino acid sequence of T. thermophilus (Tth), E. coli (Eco), M. luteus (Mlu), S. pneumoniae (Spn), and N.
gonorrhoeae (Ngo) UvrB proteins, and the alignment of
them. Black and shadowing indicate identical and
homologous (R/K, D/E, S/T, or L/I/V/F/Y/M) amino acid residues,
respectively. The deleted amino acid residues in each protein are
indicated by bars. The regions used for PCR primers,
Walker's A-type motif, and helicase core sequence are indicated
by arrows, #, and %, respectively. Predicted secondary
structures of -helix,
-sheet, and turn are indicated by a, b, and t, respectively. The
-helical
region, residues 251-295 in T. thermophilus, was underlined (see text for details).
The nucleotide
sequence of the T. thermophilus uvrB gene was determined, as
described under ``Materials and Methods,'' and its G+C
content was 68%, which agrees well with that of 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()leads support to this
possibility.
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.
Figure 3: Phylogenetic tree of the UvrB proteins and DNA/RNA helicases. The tree was constructed for the entire regions of these proteins by the neighbor-joining method (34) using the distance values calculated according to Zuckerkandl and Pauling(33) . The distance values used to construct the tree are shown on the figure. Abbreviations are as follows: Eco, E. coli; Ngo, N. gonorrhoeae; Spn, S. pneumoniae; Mlu, M. luteus; Tth, T. thermophilus; Sc-Rad3, S. cerevisiae Rad3; h-XP-D, human XP-D; Eco-SrmB, E. coli SrmB; Sc-p68, S. cerevisiae p68; mur-PL10, murine PL10; h-eIF-4AI, human eIF-4AI.
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.
Figure 5:
Far-UV (left) and near-UV (right) CD spectra of T.
thermophilus UvrB protein. Measurements were performed at 25
°C in a buffer solution containing 20 mM Tris-HCl (pH 7.5)
and 2 µM (far-UV range) or 20 µM (near-UV
range) T. thermophilus UvrB protein. The residue molar
ellipticity [] is defined as described under
``Materials and Methods.''
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. 2were 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.
Figure 6:
Temperature dependence of the residue
molar ellipticity [] at 222 nm. The heating rate was 1
°C/min. The conditions except for temperature were the same as
those described in Fig. 5.
Next,
the effect of pH on T. thermophilus UvrB protein stability was
studied on the basis of []
. 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
[
]
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, 50, 51, 52) . (
)Although this stability at high pH values is a common
feature of thermophilic proteins, the reason for it is not clear.
Figure 7:
pH-dependent changes in residue molar
ellipticity [] at 222 nm. Measurements were performed
at 25 °C after incubation of 2 µMT. thermophilus UvrB protein with each buffer for 16 h. The buffers used were: 70
mM HCl (pH 1.13), 20 mM glycine-HCl (pH 2.40), 20
mM sodium acetate (pH 3.66-3.88), 20 mM sodium
phosphate (pH 6.05-7.29), 20 mM Tris-HCl (pH
8.20-9.16), 20 mM sodium borate (pH 9.44), and 20 mM sodium carbonate (pH 10.95).
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.
Figure 9: A, kinetics of ATP hydrolysis by T. thermophilus UvrB protein at 65 °C. The amount (%) of ADP formed was plotted against the reaction time (min). ATPase assays were performed as described under ``Materials and Methods.'' Closed circles, 5 µMT. thermophilus UvrB protein, 200 µM poly(dC), and 1 mM ATP; open circles, without poly(dC); open squares, without protein and poly(dC). B, ATP concentration dependence on ATPase activity of T. thermophilus UvrB protein. The reaction mixtures contained 5 µMT. thermophilus UvrB protein and 0 (open circles) or 200 µM (closed circles) poly(dC).
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
UvrAB-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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49912[GenBank].