(Received for publication, November 9, 1994; and in revised form, January 10, 1995)
From the
UvrB plays a central role in (A)BC excinuclease. To identify the regions of UvrB which are involved in interacting with UvrA, UvrC, and DNA, deletion mutants, point mutants, and various fusion forms of UvrB were constructed and characterized. We found that the region encompassing amino acid residues 115-250 of UvrB binds to UvrA, while the region encompassing amino acid residues 547-673 binds to both UvrA and UvrC. In addition, the region between these two domains, which contains the helicase motifs II-VI, was found to be involved in binding to DNA. Within this DNA-binding region, two point mutants, E265A and E338A, were found to be unable to bind DNA while two residues, Phe-365 and Phe-496, were identified to interact with DNA. Furthermore, fluorescence quenching studies with mutants F365W and F496W and repair of thymine cyclobutane dimers by photoinduced electron transfer by these mutants suggest that residues Phe-365 and Phe-496 interact with DNA most likely through stacking interactions.
In Escherichia coli, nucleotide excision repair is
initiated by the (A)BC excinuclease which excises a wide variety of DNA
damages in a dodecanucleotide (Sancar and Tang, 1993; Grossman and
Thiagalingam, 1993). This activity results from the coordinated actions
of the UvrA, UvrB, and UvrC proteins. Specifically, UvrA is a damage
recognition protein as well as a molecular matchmaker (Sancar and
Hearst, 1993); it forms an AB
complex with UvrB
and guides UvrB to a lesion in the DNA. Upon binding to the lesion, the
DNA is bent by approximately 130° (Shi et al., 1992) and
the area around the lesion is unwound by about 5 bp (
)(Lin et al., 1992; Visse et al., 1994b). UvrA must then
dissociate from the lesion before UvrC can bind to the UvrB-DNA complex
to induce the dual incisions (Orren and Sancar, 1989, 1990; Visse et al., 1992). Upon binding of UvrC to the UvrB-DNA complex,
UvrB makes the 3` incision after which the 5` incision is made by UvrC
(Lin et al., 1992; Lin and Sancar, 1992). As evident from the
ongoing presentation, UvrB is the central component of the entire
excision repair process; it must interact with UvrA, bind specifically
to damaged DNA, interact with UvrC, make the 3` incision, and following
the dual incisions, it must then interact with both helicase II and
polymerase I to complete the repair process (Orren et al.,
1992). Despite its central role in excision repair, there is only a
limited information on the structure-function relationship of UvrB.
In the present study, we have investigated the structural basis for the multiple interactions of UvrB during nucleotide excision repair. By assaying deletion mutants, point mutants, and various fusion forms of this protein for protein-protein interaction, DNA binding, and excision activities, the UvrA, UvrC, and DNA-binding domains of UvrB have been identified.
Fusions of UvrB with the maltose-binding protein (MBP) were constructed by ligating different fragments of the uvrB gene into the pMAL-c2 expression vector (New England Biolabs). Briefly, a derivative of pUNC211, called pUNC211a, was constructed which introduced the restriction site for HincII, GTTAAC, at position 747 and a restriction site for HindIII, AAGCTT, at position 2072 of the uvrB gene (Arikan et al., 1986). Afterwards, three different fusion proteins of UvrB to MBP were constructed: MBP/UvrB(115-250), corresponding to amino acid residues 115-250 of UvrB, was made by cloning the HincII fragment of pUNC211a from positions 342 to 750 into the XmnI site of pMal-c2. MBP/UvrB(251-547), corresponding to residues 251-546 of UvrB, was made by cloning the HincII-EcoRV fragment of pUNC211a from positions 751 to 1783 into the XmnI site of pMal-c2. MBP/UvrB(547-673), corresponding to residues 547-673 of UvrB, was made by cloning the EcoRV-HindIII fragment of pUNC211a from positions 1784 to 2075 into the XmnI/HindIII site of pMal-c2. Fusion constructs were identified by restriction enzyme digestions.
The fusion proteins
were purified as follows. Single colonies of DR153 harboring the
appropriate plasmid were inoculated in 500 ml of LB with the required
antibiotics and grown at 30 °C until an O.D. of 0.6 was reached.
The culture was then induced with 0.1 mM isopropyl-1-thio--D-galactopyranoside and grown for
an additional 4 h. Afterwards cells were harvested and resuspended in
10 ml of lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 10% sucrose) per liter of culture.
Cells were lysed by sonicating 10
for 10 s each with a Branson
Sonicator. The lysate was spun for 1 h at 40,000
g at
4 °C and the supernatant was loaded onto an amylose column
equilibrated with Buffer B (100 mM Tris, pH 7.5, 1 mM EDTA, 10 mM
-mercaptoethanol, 20% glycerol) +
0.1 M KCl. The column was then washed with 5 column volume of
Buffer B + 0.1 M KCl. Bound proteins were then eluted
with 1 column volume of Buffer B + 0.1 M KCl + 10
mM maltose. Purified proteins were dialyzed and stored in
storage buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1
mM EDTA, 5 mM dithiothreitol, and 50% glycerol).
Since fusion proteins were used for most of the studies to identify
structural domains in UvrB it was essential to establish that these
proteins were ``well behaved'' with regard to their
solubility and quaternary structures. All of the constructs used were
as soluble as the wild-type protein and those that were poorly soluble (e.g. MBP/UvrB(114-120) and
MBP/UvrB(
207-225) were only characterized genetically. In
addition, the constructs used for biochemical studies behaved as
monomers as determined by gel permeation chromatography under
conditions employed to determine the oligomerization status of the
wild-type protein. Thus, we conclude that the interactions we detect
for the fusion constructs are intrinsic to the UvrB part of the fusions
and are not compounded by competing interactions that arose from newly
created protein interfaces.
The
psoralen and cisplatin containing oligomers were kindly provided by
Drs. John E. Hearst (University of California, Berkeley) and Stephen J.
Lippard (Massachusetts Institute of Technology). Oligomers without a
modified base or with a synthetic AP site were obtained from Operon
Biotechnologies. Binding assays of UvrB to these oligonucleotides were
done as follows. 1 nMP-labeled oligomers were
incubated with increasing concentrations of UvrB at 22 °C for 20
min in 50 µl of ABC buffer (50 mM Tris, pH 7.5, 10 mM MgCl
, 0.1 M KCl, 1 mM dithiothreitol, and 10% glycerol). The reaction mixtures were then
loaded onto a 10% polyacrylamide gel and electrophoresed at 80 V for 5
h at 4 °C. Binding of UvrB to DNA was then quantified by AMBIS
scanning. Since the binding of UvrB to DNA is of low affinity, the
bound fraction does not always yield a sharp band; in fact, quite often
it appears as a smear above the free (unbound) DNA. Thus, the fraction
of DNA not in the band corresponding to free DNA was considered bound
in our analysis.
Figure 1:
A, binding of UvrB
protein to UvrC affinity column. Cell-free extract from 30 ml of
DR153/pUNC211 culture overexpressing the UvrB protein was loaded onto
an UvrC affinity column (0.5 ml) containing 3 mg of UvrC protein. The
column was washed with 10 column volumes of binding buffer + 0.1 M KCl and bound fractions were eluted with a continuous
gradient of binding buffer + 0.1 M KCl to binding buffer
+ 1.0 M KCl. Fractions of 0.25 ml were collected and
analyzed by SDS-PAGE gels and silver staining. L, load; M, purified UvrB as a marker; ,
`, large subunits of
RNA polymerase. B, binding of UvrC protein to UvrB affinity
column. Cell-free extract from 1.2 ml of DR1984/pUNC3274 culture
overexpressing the UvrC protein was loaded onto an UvrB affinity column
(0.4 ml) containing 5 mg of UvrB protein. The column was washed and
bound proteins were eluted and analyzed as in panel
A.
Figure 2: A, binding of MBP/UvrB(115-250) to UvrA and UvrC affinity columns. 1.0 µg of MBP/UvrB(115-250) protein was loaded onto either an UvrA (top panel) or an UvrC (bottom panel) affinity column. The column was washed with 10 column volumes of binding buffer + 0.1 M KCl and bound fractions were eluted with binding buffer + 0.5 M KCl. Fractions of 0.25 ml were collected and analyzed by SDS-PAGE gels and silver staining. L, load; M, purified UvrC as a marker. B, binding of MBP/UvrB(547-673) to UvrA and UvrC affinity columns. 1.0 µg of MBP/UvrB(547-673) was loaded onto either an UvrA (top) or an UvrC (bottom) affinity column. The column was washed and bound proteins were eluted and analyzed as in panel A.
Figure 3: A, binding of UvrB protein to ssDNA containing a lesion. 5`-labeled substrates (1 nM) were incubated in a 50-µl reaction mixture containing increasing concentrations of UvrB in ABC buffer for 20 min at 22 °C. Samples were then loaded onto a 10% ployacrylamide gel and electrophoresed at 80 V for 5 h at 4 °C. The specific activities of the substrates were as follows: T<>HMT, 14.8 Ci/nmol; Pt-(GpG), 18.6 Ci/nmol; ABPD, 7.4 Ci/nmol; and unmodified (UM), 7.6 Ci/nmol. F, free DNA; B, bound DNA band. The UvrB concentrations in the reaction mixtures were: Lanes 1 and 6, 0 µM; lanes 2 and 7, 1.5 µM; lanes 3 and 8, 3.2 µM; lanes 4 and 9, 6.5 µM; and lanes 5 and 10, 13 µM. B, quantitative analysis of the binding data. The percent binding was determined by quantification of the radioactivity in the unbound fractions. Data from panel A and two other experiments conducted under identical conditions were averaged. Error bars show standard deviation. Square, T<>HMT; circle, Pt-(GpG); triangle, APBD; diamond, unmodified.
To determine if UvrB also bound specifically to double-strand DNA with a lesion, gel retardation assays with single- and double-stranded DNAs containing a thymine-psoralen monoadduct were conducted. Fig. 4shows that UvrB binds to ssDNA with T<>HMT but has no affinity for dsDNA containing the same adduct. Furthermore, it also appears that UvrB cannot discriminate between ssDNA and dsDNA when the DNA has no lesion (Fig. 5), which further supports the notion that UvrB binds specifically only to ssDNA with a lesion.
Figure 4: A, binding of UvrB protein to ssDNA and dsDNA containing a thymine-psoralen monoadduct. 5` labeled single- and double-strand DNA containing a thymine-psoralen monoadduct were incubated in a 50-µl reaction mixture containing increasing concentrations of UvrB in ABC buffer for 20 min at 22 °C. Samples were then loaded onto a 10% polyacryamide gel and electrophoresed at 80 V for 5 h at 4 °C. F, free DNA; B, bound DNA band. The UvrB concentrations in the reaction mixtures were: Lanes 1 and 6, 0 µM; lanes 2 and 7, 1.5 µM; lanes 3 and 8, 3.2 µM; lanes 4 and 9, 6.5 µM; and lanes 5 and 10, 13 µM. B, quantiative analysis of data in panel A. The percentage of binding was determined by quantification of the radioactivity in the unbound fractions from panel A. Square, ssDNA-T<>HMT; diamond, dsDNA-T<>HMT.
Figure 5: A, binding of UvrB protein to single-strand and double-strand oligonucleotide. 5` labeled single- and double-strand DNA were incubated in a 50-µl reaction mixture containing increasing concentrations of UvrB in ABC buffer for 20 min at 22 °C. Samples were then loaded onto a 10% polyacrylamide gel and electrophoresed at 80 V for 5 h at 4 °C. F, free DNA; B, bound DNA band. The UvrB concentrations in the reaction mixtures were: Lanes 1 and 6, 0 µM; lanes 2 and 7, 1.5 µM; lanes 3 and 8, 3.2 µM; lanes 4 and 9, 6.5 µM; and lanes 5 and 10, 13 µM. Note that the dsDNA contains some ssDNA contaminant, and a slower migrating minor species of unknown origin. B, quantitative analysis of data in panel A. The percentage of binding was determined by quantification of the radioactivity in the unbound fractions from panel A. Square, ssDNA; diamond, dsDNA.
Seven phenylalanine residues that were distributed throughout the primary structure of UvrB were replaced individually with tryptophans to obtain UvrB mutants with a Trp residue at positions 88, 107, 187, 216, 365, 496, or 527. Since the Phe to Trp substitution is a conservative change, the mutant proteins were fully functional and behaved identically to wild-type UvrB protein in every aspect including in vivo complementation. The mutant proteins were purified and the quenching of Trp fluorescence of these mutants by ssDNA was investigated.
The results of these studies are shown in Fig. 6. Several features of this data should be noted. First, the Trp emission maxima of all the mutants are between 330 and 340 nm, which is lower than the emission maximum of 355 nm for free tryptophan. The blue shift of the Trp emission typically occurs when the tryptophan is in a hydrophobic environment. Thus, it appears that all of the Trp residues and by extension, all of the Phe residues which were replaced by Trp residues, are located in a hydrophobic environment within UvrB. Second, according to fluorescence intensity, these mutants fall into three classes; F107W with the lowest intensity; F88W and F527W with intermediate intensity; and F187W, F216W, F365W, and F496W with the highest level of intensity. The environments of the various Trp residues influence the deactivation of the excited tryptophans by non-emissive pathways to different degrees and as a result, different fluorescence intensities are observed. To a first approximation, tryptophans more accessible to solvent or polar amino acids are more likely to decay by non-emissive pathway and hence, have lower quantum yield of fluorescence. Table 1shows the quantum yields of Trp fluorescence for the various UvrB mutants. Finally, ssDNA specifically quenches the fluorescence of three mutants, F187W, F365W, and F496W. Quenching can be caused by either direct contact of the DNA with these tryptophans or as a result of a conformational change that may have occurred upon DNA binding which affects the solvent accessibility to these residues. In order to differentiate between these two possibilities, we conducted photoinduced dimer splitting by wild-type and mutant proteins.
Figure 6:
Fluorescence quenching of UvrB-Trp
mutants. 3 µM mutant UvrB protein was incubated with 130
µM (in nucleotides) calf-thymus DNA (ssDNA) in 500 µl
of ABC buffer. Fluorescence emission spectra were measured with
excitation at 295 nm. A and B indicates fluorescence
emission before and after the addition of DNA, respectively. The diagram in the bottom represents the primary sequence
of the UvrB protein, the location of the helicase domains I-VI (boxes), and the sites of the F W substitutions. The
slit width was adjusted for each mutant to obtain an optimal signal,
hence the relative intensities in different panels are not directly
comparable.
Wild-type and mutant UvrB proteins were mixed with a 49-nucleotide long single-strand DNA with a centrally located T<>T within the MseI recognition site T<>TAA (Svoboda et al., 1993) and irradiated with 293 nm light. Fig. 7shows that none of the mutants which did not display any fluorescence quenching could photoreverse the dimer. Of the three mutants which displayed fluorescence quenching, F365W and F496W were capable of repairing the dimer, while F187W was not. We conclude that the quenching of fluorescence observed with F365W and F496W is the result of direct contact of the tryptophans in these mutants with the DNA and hence, these residues must be located within the DNA-binding domain of UvrB. In contrast, repeated attempts to repair dimers with F187W failed even though this mutant was quenched by DNA twice as efficiently as the other two mutants. It appears that upon binding to DNA, UvrB undergoes a significant conformational change such that residue 187 becomes substantially exposed to the solvent leading to drastic quenching of fluorescence of F187W.
Figure 7:
The repair of thymine dimer by mutant UvrB
proteins. A single-stranded 49-mer (1 nM) containing a
centrally located T<>T was incubated with 5 µM mutant UvrB protein in 250 µl of ABC buffer and irradiated
with 293 nm light at a fluence rate of 500 microwatts/cm for 30 min. After irradiation, the DNA was extracted with
phenol/chloroform, annealed with the complementary strand, and treated
with MseI restriction endonuclease, which digests only the
repaired DNA, to generate the 21-mer. This is an autoradiograph of a
10% polyacrylamide gel.
Figure 8:
Psoralen-mediated cross-linking of mutant
UvrB proteins. 1 nM terminal labeled psoralen adducted 13-mer
oligonucleotide was incubated with 10 µM UvrA, UvrC
proteins, or wild-type or mutant UvrB proteins in 30 µl of ABC
buffer for 20 min at 22 °C. The mixture was irradiated with 366 nm
light at a fluence rate of 3 milliwatts/cm for 30 min at 4
°C. The products were analyzed on 15% SDS-PAGE followed by
autoradiography.
The results are shown in Fig. 9. Three mutants, E98A, E265A, and E338A had no detectable affinity for DNA; D510A had lower affinity, and D478A had affinity for DNA comparable to that of wild-type. The discrepancy between the earlier conclusion that D510A is a DNA binding mutant (Lin et al., 1992) and the current result can be reconciled by assuming that the main defect in this mutant is in the loading step (which is what was measured previously) rather than formation of a complex with the single-stranded region of the DNA after the loading reaction. In contrast, the three mutants, E98A, E265A, and E338A, are true DNA binding mutants which are defective in maintaining a stable complex subsequent to loading. In agreement with this conclusion, it was not possible to isolate UvrB-DNA complexes of these mutants by gel exclusion chromatography (data not shown), suggesting that these mutants either have no affinity for DNA or bind DNA very transiently.
Figure 9: Binding of mutant UvrB protein to oligonucleotide containing a thymine-psoralen monoadduct. 5` labeled ssDNA (1 nM) containing a T<>HMT was incubated in 25 µl of ABC buffer containing wild-type or mutant UvrB proteins at the indicated concentrations for 20 min at 22 °C. Samples were then loaded onto a native 10% polyacrylamide gel and electrophoresed at 80 V for 5 h at 4 °C. A, autoradiographs of representative gels. WT or mutant UvrB concentrations were as follows: lanes 1 and 6, 0 µM; lanes 2 and 7, 1.25 µM; lanes 3 and 8, 2.5 µM; lanes 4 and 9, 6 µM; and lanes 5 and 10, 12.5 µM. B, quantitative analysis of the binding data. Data points from three experiments were averaged. The standard errors for all of the data points were less than 10% and hence error bars are not shown for clarity. Square, wild-type; diamond, D510A; circle, D478A; triangle, E98A; closed square, E265A; and closed triangle, E338A.
To investigate the latter point, incision assays were performed with (A)BC excinuclease reconstituted with the mutant proteins. The results are shown in Fig. 10. Unlike wild-type UvrB, only the 3` incision was made by the (A)BC excinuclease reconstituted with the E98A, E265A, and E338A mutants. Since all available data suggest that UvrC makes the 5` incision (Lin and Sancar, 1992) and that the 5` incision, under certain conditions, is the rate-limiting step (Visse et al., 1994a), these results suggest that a transient UvrB-DNA complex is formed with the mutant UvrBs and when UvrC interacts with this complex, UvrB is able to make the 3` incision. However, it also appears that these complexes are so transient that UvrB dissociates from the DNA before UvrC can make the 5` incision.
Figure 10: Incision of a 137-mer duplex with a centrally located furanside thymine-psoralen monoadduct by (A)BC excinuclease reconstituted with wild-type and mutant UvrB proteins. 1 nM substrate labeled at both termini of the damaged strand was incubated in 25 µl of ABC buffer + 2 mM ATP containing 5 nM UvrA, 80 nM wild type or mutant UvrB, 40 nM UvrC, and 0.1 µg of pBR322 for 20 min at 37 °C. The reaction was stopped by adding 2 µl of a 1:1 mixture of 0.25 M EDTA, 5 mg/ml oyster glycogen, and 60 µl of EtOH. The precipitate was collected by centrifugation, resuspended in formamide/dye mixture, and analyzed on a 8% polyacrylamide DNA sequencing gel. The products generated by the dual incisions are a 66-mer indicated by 5` incision, and a 60-mer indicated by 3` incision, respectively. The band marked 3` uncoupled is a 78-mer which can only be generated when the enzyme makes the incision 3` to the lesion but fails to make the incision 5` to the damage (Lin and Sancar, 1992).
UvrB plays a central role in nucleotide excision repair. It interacts with both the UvrA and UvrC proteins in addition to binding, bending, and incising DNA. In this work, we have attempted to identify the regions of UvrB involved in interacting with UvrA, UvrC, and DNA. In light of our findings, the following structure-function model for UvrB is proposed (Fig. 11).
Figure 11: Structure-function model for UvrB. The black boxes numbered with Roman numerals are the so-called helicase motifs. UvrA binds to two well defined domains in the amino- and carboxyl-terminal halves of the protein. The UvrA-binding region in the amino-terminal domain is homologous to the UvrA-binding region of TRCF. The UvrA-binding region in the carboxyl-terminal domain overlaps the UvrC-binding site. The helicase motifs participate in DNA binding and within this DNA-binding domain, residues Glu-265, Glu-338, and Asp-510 contact DNA possibly through salt bridges with phosphates counterions while residues Phe-365 and Phe-496 presumably intercalate into the DNA. The carboxyl-terminal 43 amino acids contain the catalytic residue Glu-639 (Lin and Sancar, 1992).
There are two regions of UvrB
which are responsible for binding to UvrA. The first region,
encompassing amino acid residues 115-250, is homologous to a
region in the amino-terminal half of TRCF which also binds UvrA (Selby
and Sancar, 1993). The second region, encompassing amino acid residues
547-673, is located in the carboxyl terminus of UvrB. Previous
studies have shown that cleavage of the 43 carboxyl-terminal amino
acids of UvrB generates UvrB (Arikan et al.,
1986), which is catalytically inactive (Lin et al., 1992) but
binds both UvrA (Orren and Sancar, 1989) and UvrC (Lin et al.,
1992) with normal affinity. Therefore, the carboxyl-terminal region
that is involved in binding to UvrA can be narrowed down to amino acid
residues 547-630.
Recently, the 135-amino acid long region in
the TRCF, which has 25% sequence identity with amino acids
115-250 of UvrB, was found to be sufficient for high affinity
binding of TRCF to UvrA (Selby and Sancar, 1995). Since UvrB makes a
tighter complex with UvrA compared to TRCF, it is possible that the
region in the carboxyl-terminal half of UvrB, which also interacts with
UvrA, contributes to the formation of a more stable UvrA-UvrB complex.
Significantly, the carboxyl-terminal domain of UvrB which interacts
with UvrA is also essential for formation of a UvrB-UvrC complex. This
finding, therefore, explains why a ternary complex containing all three
subunits cannot be isolated, and why UvrA must dissociate from the
AB
-DNA complex before UvrC can bind to the
UvrB-DNA complex and initiate the dual incisions.
UvrB is a member
of a family of proteins which require the assistance of a molecular
matchmaker to bind DNA (Sancar and Hearst, 1993). In the case of UvrB,
UvrA is required to load UvrB onto DNA with a lesion. It has been
suggested that many of the proteins which bind to DNA by this mechanism
can actually form a DNA-protein complex in the absence of the
matchmaker under special conditions such as extremely high protein
concentrations or the inclusion of macromolecular crowding compounds in
the reaction mixture. For example, the -clamp of DNA Pol III which
is loaded onto DNA by the
complex (Kuriyan and O'Donnell,
1993) can associate with DNA directly when a 100-fold molar excess over
the DNA Pol III holoenzyme of the
subunit are used in the
reaction (Crute et al., 1983; Kwon-Shin et al.,
1987). Similarly, the gp45 protein, which is the polymerase clamp for
T4 DNA polymerase (gp43) and is loaded onto DNA by the gp44/62 complex
(Huang et al., 1981; Nossal and Alberts, 1984), can be loaded
directly onto DNA by using either a high concentration of gp45 (Reddy et al., 1993) or by including polyethylene glycol in the
reaction mixture (Sander et al., 1994). Here we show that this
property of proteins which use the aid of molecular matchmakers also
applies to UvrB. Specifically, we demonstrate that at high
concentrations of UvrB, the protein can bind to ssDNA with a lesion in
the absence of UvrA. Furthermore, based on the limited number of
lesions we tested, it appears as though the affinity of UvrB to a
lesion correlates with how efficiently that lesion is excised by the
(A)BC excinuclease.
Upon loading UvrB to a lesion, UvrA dissociates from the DNA, leaving a stable UvrB-DNA complex that is bent by about 130 °C and in addition, is locally denatured by about 5-6 bp around the lesion (Sancar and Tang, 1993). This suggests that UvrB is the ``ultimate damage recognition subunit'' of the (A)BC excinuclease which determines the efficiency of removal of a lesion from DNA. Hence, this study proposes that in contrast to UvrA (the proximal damage recognition subunit), which recognizes damage in a duplex, UvrB binds to the area of denaturation around the lesion to form a UvrB-DNA complex and the formation of this complex then determines whether or not incision occurs.
It has also been reported previously that although the (A)BC excinuclease recognizes a wide variety of damages, the level of excision is widely different for each type of damage (Huang et al., 1994). This study now shows that the affinity of UvrB to a particular lesion is correlated with how efficiently that lesion is excised and thus further supports the notion that UvrB is the ultimate damage recognition subunit of the (A)BC excinuclease.
This conclusion, then, raises the question of how UvrB binds to damaged ssDNA. Several observations bear on this question. First, mutations in the charged residues of the so-called helicase motifs interfere with binding (Lin et al., 1992; Moolenaar et al., 1994; Seeley and Grossman, 1990) suggesting that these motifs constitute at least part of the DNA-binding domain. Second, these studies suggest that ionic interaction play an important role in the formation of a UvrB-DNA complex. However, seemingly parodoxically, the UvrB-DNA complexes are very stable in high-ionic strength buffers (Orren and Sancar, 1989, 1990) suggesting that the main interaction between UvrB and DNA is hydrophobic. Finally, in this study we have demonstrated by fluorescence quenching and photoinduced electron transfer that residues Phe-365 and Phe-496 of UvrB are presumably in direct contact with the DNA bases. All these observations combined suggest the following model for the formation of UvrB-DNA complexes: the initial contact with the DNA which is aided by UvrA, is mainly ionic in nature. Afterwards, upon ATP hydrolysis, UvrB undergoes a conformational change which expose residues Phe-365, Phe-496, and possibly other aromatic residues for direct interaction, perhaps by intercalation, with the bases.
The proposed mode of interaction may also explain the ``specific'' binding of UvrB to a lesion. Since the (A)BC excinuclease repairs virtually all lesions in DNA, previously we and others have argued that the subunits of the enzyme cannot be making direct contact with an essentially infinite number of chemical groups which constitute a ``lesion'' and hence it has been generally assumed that the enzyme recognizes the backbone distortions of the DNA (Sancar and Tang, 1993; Grossman and Thiagalingam, 1993). In light of our finding of direct binding of UvrB to lesions, we would like to propose the following model: UvrB in fact does have a lesion binding pocket where the modified bases fit in. We propose that this is a hydrophobic pocket and because of lack of requirements for specific H-bond donors or acceptors or for formation of salt bridges of unique orientations, a vast number of chemical groups can be accommodated within this pocket. Presumably the degree of hydrophobicity, the size as well as some other, as yet to be determined, factors contribute to the relative affinities of various side groups for UvrB and hence their susceptibilities to function as substrates.