(Received for publication, July 5, 1995; and in revised form, September 25, 1995)
From the
The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion. The incisions are in an ordered fashion, first on the 3`-side and next on the 5`-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex. In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3`-incision. The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil. We therefore propose that UvrB and UvrC interact through the formation of such a structure. The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain. Binding of UvrC to the C-terminal region of UvrB is not important for the 5`-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required. Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3`-incision. This region, however, contains the residue (Glu-640) that was proposed to be involved in 3`-catalysis, since a mutation of the residue (E640A) fails to promote 3`-incision (Lin, J. J., Phillips, A. M., Hearst, J. E., and Sancar, A.(1992) J. Biol. Chem. 267, 17693-17700). We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo. As a consequence of these results, it is still an open question as to whether the catalytic site for 3`-incision is located in UvrB, in UvrC, or is formed by both proteins.
The UvrB protein is a key subunit of the UvrABC endonuclease
from Escherichia coli involved in the repair of a wide range
of different types of DNA damage (for reviews, see (1, 2, 3) ). Together with UvrA, UvrB forms a
trimeric UvrAB complex, which is able to recognize a lesion
in the DNA. Upon binding to the lesion, the helicase activity of the
UvrA
B complex results in the loading of the UvrB on the DNA (4) and the subsequent release of the UvrA protein(5) .
The DNA in the resulting UvrB-DNA preincision complex is unwound (6) and kinked(7) , and the UvrB protein seems to be
bound on the opposite side of the DNA helix to the site of the
damage(8) . Upon binding of UvrC to the preincision complex,
the DNA is incised on both sides of the lesion. First, the 3`-incision
is made (9) at the 4th or 5th phosphodiester bond downstream
from the lesion, followed by the 5`-incision at the 8th phosphodiester
bond upstream from the lesion(10, 11) . When UvrA is
still present in the preincision complex, this can lead to an
additional incision at the 15th phosphodiester bond 5` of the lesion (11) . The oligonucleotide containing the damage is released by
the action of UvrD, and the single-stranded gap is filled in by DNA
Polymerase I(12) . The repair reaction is completed with the
closure of the remaining nick by DNA ligase.
Although the overall repair mechanism of the UvrABC system is quite well understood, little is known about how the Uvr proteins interact with each other or with the damaged DNA. The dimerization domain of the UvrA protein has been shown to be located in the N-terminal 605 amino acids(13) , but the domain for interaction with UvrB has not yet been identified. It has been shown that the C-terminal 314 amino acids of UvrC are sufficient to support both incisions, albeit less efficiently than the complete protein(14) . Mutations at four different positions in the protein (Asp-399, Asp-438, Asp-466, and His-538) were shown to inhibit the 5`-incision but not the 3`-incision, indicating that these residues might form the catalytic site for the 5`-incision(9) . The UvrC domain involved in interaction with the UvrB-DNA preincision complex is not known, but two small areas of homology with UvrB can be identified in the UvrC protein (residues 197-210 and 326-331), which might be implicated in this function. The UvrB protein contains a relatively large domain of homology (residues 114-251) with the transcription-repair coupling factor, which is the product of the mfd gene(15) . Since transcription repair coupling factor is able to bind UvrA, it has been postulated that the homologous domain in UvrB also interacts with UvrA(15) .
In this paper, we show that the domain of UvrB that is involved in the interaction with UvrC after formation of the preincision complex is located in the C-terminal region of the protein. Since the postulated catalytic residue for 3`-incision (Glu-640, (16) ) is also located in this C-terminal region, we have isolated a mutant containing a mutation (E640A) of this residue and have tested UvrC binding. Surprisingly, however, in our hands, this mutant resulted in normal UvrC binding and incision in vitro and normal survival after UV irradiation in vivo.
The 96-bp ()DNA fragment containing a single cis-Pt
GG adduct (Fig. 8) was constructed from six
oligonucleotide fragments as described(11) . Construction of
the DNA substrate containing a nick on the 3`-side of the lesion was
carried out using the same oligonucleotide fragments, but the
3`-oligonucleotide in the upper strand was not phosphorylated to
prevent ligation (see Fig. 8).
Figure 8:
Synthesis of DNA substrate containing a
nick on the 3`-side of the damage. The 96-bp DNA fragment containing a
single cis-PtGG adduct was constructed by ligation of
six phosphorylated oligonucleotide fragments as described(11) .
For the construction of the DNA substrate containing a nick on the
3`-side of the lesion, the same oligonucleotide fragments were used,
but the 3`-oligonucleotide in the upper strand was not phosphorylated
to prevent ligation. Note that the 3`-nick is shifted three
phosphodiester bonds with respect to the 3`-incision position, which is
indicated with an arrow.
Figure 1:
Formation of a specific UvrBC-DNA
complex. A, the cis-PtGG-containing fragment
was incubated with 2.5 nM UvrA and 100 nM UvrB with
or without 20 nM UvrC. After allowing protein binding for 20
min at 37 °C, the mixture was postincubated with UvrA preimmune
serum (oA), UvrA antiserum (
A), UvrB preimmune
serum (oB), UvrB antiserum (
B), or UvrC
antiserum (
C) for 5 min on ice. The positions of the
UvrA
B-, UvrB-, and UvrBC-DNA complexes are indicated.
Complexes that react with the antisera either move into the gel with a
slower mobility (lanes 3, 4, and 8) or no
longer enter the gel and remain in the slot (lanes 2, 3, 6, and 8). Note that the UvrC antiserum
gives a small amount of cross-reactivity with the UvrB protein (lane 4). B, the cis-Pt
GG-containing
fragment was incubated with 10 nM UvrA, 20 nM UvrC,
and 100 nM wt UvrB (lanes 1 and 2) or mutant
UvrB (lanes 3-8), after which UvrC preimmune serum (oC) or UvrC antiserum (
C) was added. The
positions of the UvrA
B- and UvrBC-DNA complexes are
indicated. The UvrBC-DNA complexes that are bound by UvrC antiserum
remain in the slot of the gel.
Figure 2: Coiled-coils plot of the UvrB protein. The amino acid sequence of the UvrB protein was analyzed with the program COILS(21) . The probability of coiled-coil formation is plotted against the residue position.
Figure 3: Alignment of the C-terminal regions of different UvrB proteins and the homologous regions of different UvrC proteins. The C-terminal regions of the UvrB proteins from E. coli (Ec-B, (24) ), Neisseria gonorhoeae (Ng-B, (34) ), Streptococcus pneumoniae (Sp-B, (35) ), Micrococcus luteus (Ml-B, (36) ), and Zymomonas mobilis (Zm-B, K. K. H. Reuter and R. Ficner, unpublished data) are aligned with the homologous regions of the UvrC proteins from E. coli (Ec-C, (37) ), Pseudomonas fluorescens (Pf-C, (38) ), and Bacillus subtilis (Bs-C, (39) ). The conserved amino acids are in bold. The heptad repeats that are characteristic for the formation of a coiled-coil motif (abcdefg), with hydrophobic amino acids at the a and d positions are indicated. The end points of the truncated E. coli UvrB proteins used, the position of the two possible OmpT cleavage sites leading to UvrB* as determined in this paper, and the position of the mutation E640A are shown.
Figure 4: Size comparison of UvrB* and UvrB630. Wild type UvrB (lane 1), UvrB* (lane 2), and UvrB630 (lane 3) were loaded on a protein gel, after which they were visualized by ECL-Western blotting.
Both the UvrB* and UvrB630
proteins were tested for their ability to form UvrBC-DNA complexes in
the mobility shift assay with UvrC antibodies. Fig. 5(lanes
1-4) shows that neither of the two truncated UvrB proteins
is capable of forming such a complex. DNase I footprinting of the
mutant UvrAB complexes on a DNA fragment containing a cis-Pt
GG adduct confirmed that both truncated proteins
do form normal preincision complexes (results not shown), as was shown
before for the UvrB* protein(5, 23) . Therefore, we
conclude that the C-terminal 43 amino acids of UvrB contain an
important determinant for UvrC binding to the preincision complex.
Figure 5:
UvrC binding in the presence of truncated
UvrB proteins. The cis-PtGG-containing fragment was
incubated with 10 nM UvrA, 20 nM UvrC, and 100 nM of wt UvrB (lanes 1, 3, and 5), UvrB* (lane 2), UvrB630 (lane 4), or UvrB649 (lane
6), after which UvrC antiserum was added. The positions of the
UvrA
B- and UvrB-DNA complexes are indicated. The UvrBC-DNA
complexes that are bound by UvrC antiserum remain in the slot of the
gel.
Figure 6:
UvrABC
incision of the cis-PtGG-containing fragment in the
presence of the truncated UvrB proteins. The 96-bp cis-Pt
GG-containing fragment was 5`-labeled in the
damaged strand. The DNA was incubated with or without 10 nM UvrA, 20 or 40 nM UvrC, and 100 nM wild type (wt) or truncated UvrB. The incision products were analyzed on
a denaturing gel. The fragment of 42 nucleotides corresponds to the
products of the 5`-incision. The 35-nucleotide fragment is the result
of the extra incision at the 5`-side, which has been described
before(11) . For the wild type UvrB also a small amount of the
uncoupled 3`-incision product (54 nucleotides) is
visible.
Figure 7:
UvrABC incision of the cis-PtGG-containing fragment in the presence of UvrB
mutant E640A. The 96-bp cis-Pt
GG-containing fragment was
5`-labeled in the damaged strand. The DNA was incubated with 10 nM UvrA, 20 nM UvrC, and 100 nM wild type (wt) UvrB (lane 1) or UvrB(E640A) (lane 2).
The incision products are indicated.
Figure 9: UvrABC incision of the cis-Pt fragment containing a 3`-nick. The normal cis-Pt fragment (ds) and the same fragment containing a nick at the 3`-side of the lesion (nicked) were 5`-labeled in the damaged strand. The DNA was incubated with 10 nM UvrA and 100 nM wild type (wt) UvrB (lanes 1-6) or UvrB630 (lanes 7-9) for 10 min. Next, 20 nM UvrC was added, and the incision reaction was stopped after 1, 2.5, or 5 min by adding 77 mM EDTA. Non-incised DNA results in fragments of 96 (normal substrate) or 57 nucleotides (nicked substrate). The products of the uncoupled 3`-incision (54 nucleotides), the 5`-incision (42 nucleotides), and the extra 5`-incision (35 nucleotides) are indicated.
Figure 10:
Analysis of complexes formed on a nicked
substrate. The cis-Pt containing DNA with the 3`-nick was
incubated with 10 nM UvrA (lanes 2-4), 20
nM UvrC (lanes 3 and 4), and 100 nM wild type (wt) UvrB (lane 3) or UvrB630 (lanes 2 and 4), after which UvrC antiserum was added (lanes 3 and 4). Note that due to the procedure used
to construct the nicked fragment, single-stranded DNA (ssDNA)
is present, which migrates faster than the double-stranded fragment.
This single-stranded DNA gives rise to the formation of a
UvrAB-single-stranded DNA complex, which migrates somewhat
faster as the normal UvrA
B-DNA
complex.
We therefore propose two binding modes for the UvrC protein; for the 3`-incision, stable protein-protein interaction between UvrB and UvrC is required. This 3`-incision results in a conformational change in the complex, which is subsequently bound by another domain of UvrC, resulting in 5`-incision. The second interaction of UvrC, with the DNA and/or with another domain of UvrB, could possibly be accomplished by the UvrC molecule that is already present in the complex, but alternatively it might result from the binding of a second UvrC molecule.
The UvrB protein plays a key role in the nucleotide excision
repair by the UvrABC endonuclease. First, UvrB interacts with the UvrA
dimer, which subsequently helps it to bind to the DNA lesion where it
finally interacts with the UvrC protein to promote incision. In this
paper, we have shown that at least the 24-amino acid C-terminal region
of UvrB is required for the interaction of the preincision complex with
UvrC. One interpretation of this observation might be that the
C-terminal region of UvrB induces a conformational change in the DNA of
the preincision complex, which is subsequently recognized by UvrC.
DNase I footprint analysis of preincision complexes formed by wild type
UvrB or the truncated UvrB* protein, however, displays the same pattern
of protected and enhanced cleavage sites, indicating that the DNA
conformations in these complexes are the same (16) . ()We favor the hypothesis that the C-terminal region of UvrB
is directly involved in the binding of UvrC, since it contains homology
with a central domain of UvrC (22, 24) and forms part
of a longer region encompassing residues 629-670 that is
predicted to be able to form a coiled-coil motif (Fig. 3).
Coiled-coils are formed by two or more amphipathic
helices, which
display a pattern of hydrophilic and hydrophobic residues in a
7-residue repeat(25) . They have been found to be implicated in
protein-protein interactions such as the multimerization of myosins and
keratins (25) or the interaction between Fos and
Jun(26) . The binding of UvrC to the preincision complex might
well therefore be stabilized by the formation of such a coiled-coil
structure. Interestingly in this respect, the C-terminal regions of all
UvrB proteins identified as well as the homologous regions of all the
known UvrC proteins show the same high probability of coiled-coil
formation (Fig. 3). These regions also have in common the fact
that they contain a discontinuity in the heptad repeats. Such
discontinuities are often found in proteins with coiled-coils, and they
are thought to contribute to protein function either by creating fixed
bends or by providing flexibility to the protein
interface(27) . Flexibility of the UvrB-UvrC interacting domain
might be important for the positioning of the catalytic site of the
UvrBC complex, since it is likely that different types of damage impose
structural variation upon the UvrBC-DNA complexes. It has been shown
for cis-Pt-containing DNA that the binding of UvrC to the
preincision complex is much faster than the incision(20) ,
indicating that at least for this lesion the binding of UvrC does not
directly position the catalytic site at the scissile phosphodiester
bond.
The protein region preceding the postulated coiled-coil motif
might also contribute to flexibility of the UvrBC interacting domain.
This region is non-conserved and has a variable length in the UvrB
proteins from different organisms (Fig. 3). From the molecular
mass determination carried out on the UvrB* protein, we suggest that
the cleavage site(s) of OmpT are between Lys-607 and Ala-608 and/or
between Lys-609 and Gly-610, which is in this non-conserved region. In
addition, cleavage by a number of other proteases also results in
UvrB*-like fragments(33) . These properties
indicate that the UvrC binding domain of UvrB is connected to the rest
of the protein by a flexible hinge. Such a hinge might allow the
positioning of the active site in different preincision complexes
formed on different lesions.
The stable interaction between the C
terminus of UvrB and UvrC that can be shown in a band shift assay seems
only to occur after the preincision complex is formed. This is
illustrated by the fact that helicase mutants of UvrB that are able to
recognize the DNA lesion in the AB complex but are impaired
in preincision complex formation are also impaired in UvrC binding (Fig. 1B). Moreover, no specific UvrBC complexes can be
observed when a mixture of both proteins is run on a native gel in the
absence of DNA (results not shown). Possibly, the stable binding of the
UvrC protein not only requires interaction with UvrB but also with the
DNA, and the latter might be dependent on the specific conformation
that accompanies the formation of the preincision complex. On the other
hand, it is also possible that formation of the preincision complex is
needed to either expose the C-terminal domain of UvrB or to induce the
transition to the proper
-helical structure to allow coiled-coil
formation with UvrC. Such a ``spring-loaded'' mechanism has
also been described for the hemagglutinin protein, where conformational
changes upon membrane fusion result in the transition of a loop region
to a coiled-coil conformation(28) .
The stable interaction between UvrC and the C-terminal part of UvrB appears to be important for 3`-incision, although even without this stable interaction 1-2% incision can still occur. This low amount of incision is comparable to the amount of incision observed when a maltose binding protein-UvrC fusion protein was used, which contains only the C-terminal 314 amino acids of UvrC(14) . Since this UvrC fusion protein lacks the UvrB homology, the impaired incision in the presence of this protein might be due to the inability to stably bind the preincision complex. The residual incisions that are observed with UvrB and UvrC proteins lacking their postulated coiled-coil motif does, however, imply that in the UvrBC-DNA complex, there are additional weaker interactions between the two proteins or between UvrC and the DNA. Recently, a weak interaction between UvrB and UvrC in the absence of DNA has been demonstrated using affinity chromatography(29) . The domain of UvrB involved in this interaction could be defined as lying in the region encompassing residues 547-673. It is possible that this weak interaction between UvrB and UvrC is responsible for the residual incision that occurs when the stable interaction domain is absent. It is also conceivable that the UvrBC interaction that was observed in solution is not involved in 3`- but in 5`-incision. It was shown (16) that the UvrB* protein gives rise to a normal 5`-incision using a substrate that is pre-incised at the 3`-position. In this paper, we have shown that truncated UvrB proteins that lack up to 99 C-terminal amino acids are also capable of efficient 5`-incision of a substrate that contains a nick near the 3`-incision site. Apparently, the stable UvrBC interaction that is important for 3`-incision is not needed for the 5`-incision event. The inability to detect specific UvrBC-DNA complexes with a truncated UvrB protein in the presence of a nicked substrate in a band shift assay suggests that the UvrBC interaction involved in 5`-incision is very weak. Therefore, the observed UvrBC interaction in solution might represent the interaction that induces 5`-incision. If indeed the region from residues 547 to 673 represents a functional binding domain for 3`- and/or 5`-incision, this region can be narrowed down to residues 547-574 regarding the incision efficiency of the UvrB574 mutant.
The truncated UvrB mutants used in this study not only allow possible UvrC binding domain(s) to be assigned but they also yield information concerning the UvrA binding domain. It was shown that the UvrB* protein can still bind UvrA(5) . Here, we show that the incision efficiencies in the presence of UvrB574 and UvrB* are the same, implying that UvrB574 normally binds UvrA. This allows the UvrA binding domain to be assigned to the N-terminal 574 amino acids of the UvrB protein. This is in agreement with the earlier observation that the UvrB574 mutant has a dominant phenotype in vivo(4) .
It has been postulated that the C-terminal domain of UvrB is directly involved in catalysis of the 3`-incision(16) . This suggestion was derived from the observations that the UvrB* protein and a UvrB mutant carrying a E640A substitution are both unable to incise the DNA at the 3`-position. In this paper, we show that UvrB* has a residual 3`-incision activity (1-2%) on a cis-Pt-containing substrate, and we present evidence that the impaired incision efficiency is the result of impaired UvrC binding. Furthermore, we found that introduction of the E640A substitution in UvrB resulted in a protein with identical properties to those of the wild type protein in vivo as well as in vitro. It is not clear why our E640A mutant is different from the one isolated by Lin et al.(16) . However, the fact that the mutant protein isolated by these authors is proficient in preincision complex formation and deficient in 3`-incision does not necessarily mean that it is affected in catalytic activity since, like UvrB*, it could also be deficient in UvrC binding. In any case, since UvrB574 is still capable of promoting incision, it can be concluded that the C-terminal 99 amino acids of UvrB do not directly participate in the catalysis of phosphodiester bond cleavage. Although the active site for 3`-incision might be located in the remainder of the UvrB molecule, it seems equally possible that it is entirely located in UvrC or that it is formed at the interface between the UvrB and UvrC proteins. Such an interface could be formed by the postulated additional weak interaction between UvrB and UvrC, which might be responsible for the residual 3`-incision with the truncated UvrB proteins.
Our model for the UvrABC incision reaction can be
summarized as follows. Loading of UvrB onto the site of DNA damage
results in local unwinding of the DNA and a conformational change in
the protein that exposes the UvrC binding domain. This domain interacts
with the homologous region of UvrC through formation of a coiled-coil,
which together with possible UvrC-DNA interactions leads to a very
stable complex. In this complex the active site for 3`-incision, formed
by UvrB, UvrC, or both proteins, needs to be positioned, which, in the
case of a cis-Pt lesion is a relatively slow process.
Flexibility in the UvrBC-interacting domain might facilitate this
positioning. Introduction of a nick at the 3`-site induces a
conformational change in the UvrBC-DNA complex, which subsequently
leads to a very fast 5`-incision. Since the exact position of the
3`-nick does not seem important, the conformational change induced by
the nick might be the mere release of stress in the DNA molecule,
possibly leading to further unwinding and the generation of a double
stranded-single stranded DNA junction near the site of the 5`-incision.
Such a junction might be the determinant for 5`-incision by UvrC. A
similar mechanism has been proposed for the 5`-incision by the
Rad1Rad10 complex in the nucleotide excision repair of Saccharomyces cerevisiae(30, 31) and for the
3`-incision by XPG in human excision repair(32) . The
5`-incision might be catalyzed by the same UvrC that is already bound
to the complex and that has induced 3`-incision. Alternatively, a
second UvrC molecule might be responsible for the cleavage at the
5`-site. The fact that the stable UvrBC interaction that is involved in
3`-incision does not seem to contribute much to the efficiency of
5`-incision argues for the latter.
Addendum-After submission of this manuscript, D. S. Hsu and A. Sancar analyzed the uvrB gene of the E640A mutant that was used in the studies of Lin et al.(16) . The mutant turned out to contain a frameshift resulting in a truncated UvrB protein that lacks the C-terminal 33 amino acids, which explains that it shows the same properties with respect to 3`- and 5`-incision as the truncated UvrB proteins used in our study.