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J Biol Chem, Vol. 273, Issue 52, 34896-34903, December 25, 1998
From the Laboratory of Molecular Genetics, Leiden Institute of
Chemistry, Gorlaeus Laboratories, Leiden University,
2300 RA Leiden, The Netherlands
Incision of damaged DNA templates by UvrBC in
Escherichia coli depends on UvrA, which loads UvrB on the
site of the damage. A 50-base pair 3' prenicked DNA substrate
containing a cholesterol lesion is incised by UvrABC at two positions
5' to the lesion, the first incision at the eighth and the second at
the 15th phosphodiester bond. Analysis of a 5' prenicked cholesterol
substrate revealed that the second 5' incision is efficiently produced
by UvrBC independent of UvrA. This UvrBC incision was also found on the
same substrate without a lesion and, with an even higher efficiency, on
a DNA substrate containing a 5' single strand overhang. Incision
occurred in the presence of ATP or ADP but not in the absence of
cofactor. We could show an interaction between UvrB and UvrC in
solution and subsequent binding of this complex to the substrate with a 5' single strand overhang. Analysis of mutant UvrB and UvrC proteins revealed that the damage-independent nuclease activity requires the
protein-protein interaction domains, which are exclusively needed for
the 3' incision on damaged substrates. However, the UvrBC incision uses
the catalytic site in UvrC which makes the 5' incision on damaged DNA substrates.
In Escherichia coli
nucleotide excision repair is initiated by the UvrA, UvrB, and UvrC
proteins. Sequential action of these three proteins leads to two
incisions in the damage-containing DNA strand, one on either side of
the lesion (1-3). In solution a UvrA2B complex is formed,
in which UvrA is the damage-recognizing subunit (4). After initial
binding of the trimeric protein complex to the site of a DNA lesion,
UvrB is loaded onto the DNA, forming a stable UvrB·DNA preincision
complex (5). The actual incisions take place in a complex consisting of
DNA, UvrB, and UvrC. The first incision is made at the third, fourth,
or fifth phosphodiester bond 3' of the lesion, followed by an incision at the eighth phosphodiester bond 5' of the lesion (6). Several observations indicate that the conformation of the UvrBC·DNA complex differs for the 3' and 5' incision reactions. First of all, the 5'
incision can only take place when a nick is present at the 3' incision
position, introduced either enzymatically or artificially (7, 8). This
strongly suggests that the DNA adopts a different conformation in the
two complexes. A second observation is that the 3' incision requires
the interaction between the C-terminal domain of UvrB and a homologous
internal domain of UvrC, whereas this interaction is dispensable for
the 5' incision (8, 9). Conversely, the 5' incision requires the
presence of a DNA binding domain located in the C-terminal part of
UvrC, whereas this domain can be omitted for the 3' incision (10). In
the initial UvrBC·DNA complex the catalytic site for 3' incision is
most likely positioned at the scissile phosphodiester bond, incision of
which triggers a transition in the complex positioning the catalytic
site for the 5' incision. The catalytic site residues responsible for
3' incision have not yet been identified. They might be present in UvrB, in UvrC, or in both subunits. The catalytic site for 5' incision
seems to be entirely located in UvrC, because several acidic amino
acids in this protein were identified to be essential (11).
It has been reported for a number of different lesions that after 5'
incision, additional cutting takes place 7 nucleotides from the normal
5' incision site (12-14). Additional cutting was recently shown to be
related to a damage-independent incision activity of the UvrABC
proteins on a substrate containing a single strand-double strand
junction (15). Another damage-independent incision activity of the Uvr
proteins was reported by Zou et al. (16). This incision was
observed on a specific DNA Y substrate (comparable with substrate S1 in
Fig. 1A). In the absence of UvrA this specific DNA structure
was incised by the UvrBC proteins using either ATP or ADP. The incision
position was mapped three or four nucleotides 5' to the single
strand-double strand junction, and it was postulated that the
UvrBC·DNA complex formed on the Y substrate is structure-specific and
mimics the post-3' incision complex on a damaged substrate (16).
We have studied Uvr(A)BC incision in damaged and nondamaged DNA using a
set of defined DNA substrates of 50 base pairs. The same UvrBC nuclease
activity that incises Y substrates is responsible for very efficient
incision in undamaged double-stranded DNA molecules containing a nick
or a 5' single strand overhang and for the additional 5' incision in
damaged DNA. This damage-independent nuclease activity of UvrBC uses
the catalytic site in UvrC that is normally responsible for the 5'
incision on damaged substrates, but in addition protein domains of UvrB
and UvrC are required, which on damaged substrates are involved in 3'
incision exclusively.
Proteins and Antibodies--
The purification of the wild-type
UvrA, UvrB, and UvrC proteins (17), the UvrB* mutant (8), the UvrB
mutants G509S and R544H (18), the UvrC mutant (L221P,F223L) (9), and
UvrC554 (10) have been described. The clone encoding the active site mutant UvrC (D466A) was kindly provided by A. Sancar, and the protein
was purified according to the procedure described for the wild-type
UvrC. The polyclonal antibodies against UvrB and UvrC were raised in
rabbits as described (17).
Construction of DNA Substrates--
All oligonucleotides were
provided by Eurogentec (Seraing, Belgium). The cholesterol "lesion"
(also described in Ref. 19) is attached to a propanediol backbone
instead of a nucleoside (Fig. 1B) and was introduced into
the desired sequence by standard phosphoramidite chemistry at position
27 of the top strand. The presence of the cholesterol compound was
verified using mass spectrometry (Eurogentec) and denaturing
polyacrylamide gel electrophoresis. All "damaged" oligonucleotides
contained 100% cholesterol. The purity of the nondamaged
oligonucleotides was checked using denaturing gel electrophoresis. The
nucleotide sequence of the Y substrate is shown in Fig. 1A.
All other DNA substrates are of the sequences shown in Fig.
1B. Construction of the substrates was carried out by the
following procedure. The 5' top strand oligo (4 pmol) was terminally
labeled using T4 polynucleotide kinase (10 units) in 70 mM
Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM dithiothreitol, and 5 pmol of [ Incision Assay--
The DNA substrates (40 fmol) were incubated
with 2.5 nM UvrA, 100 nM UvrB, and 50 nM UvrC in 20 µl of Uvr-endo buffer (50 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, and 0.1 µg
BSA/µl) containing 1 mM ATP and 85 mM KCl.
The UvrBC endonuclease activity was tested using 100 nM
(mutant) UvrB and 50 nM (mutant) UvrC in 20 µl of Uvr-endo buffer containing 1 mM ADP or 1 mM ATP
and 85 mM KCl. After 60 min at 37 °C the incision
reaction was terminated by glycogen-ethanol precipitation. The
precipitated DNA was collected by centrifugation and resuspended in 10 µl of H2O. The volumes of the samples were reduced to 2 µl using a Speedvac concentrator (Savant) and 2 µl of
formamide/dyes was added. The samples were run on a 15% acrylamide gel
containing 6 M urea. Incision reactions according to
Gordienko and Rupp (15) were performed in 50 mM Tris-HCl
(pH 7.5), 10 mM MgCl2, 150 mM NaCl,
2 mM ATP, and 5 mM dithiothreitol using 100 nM UvrA, 100 nM UvrB, and 100 nM
UvrC. Incisions were quantified using a Betascope 603 blot analyzer (Betagen Corp., Waltham, MA). The incision frequencies are the averages
of at least three independent experiments.
Bandshift Assay--
The UvrBC protein-DNA complexes were formed
in Uvr-endo buffer containing 95 mM KCl without cofactor or
when indicated in the presence of either 1 mM ADP or 1 mM ATP in a total volume of 10 µl. After 5 min at
37 °C 1 µl of serum was added when indicated, or the samples were
directly loaded on a 3.5% native polyacrylamide gel in 0.5×
Tris-borate EDTA. The gel was run at room temperature at 8 mA, and the
protein-DNA complexes were visualized using autoradiography.
Visualization of UvrBC Complex on Native Gel--
2
µM (mutant) UvrB and 2 µM UvrC were
incubated in Uvr-endo buffer containing 100 mM KCl, and
after 6 min at 37 °C the incubation was loaded on a 3.5% native
polyacrylamide gel (without nucleotide cofactor). After the run the gel
was bound to a Whatman No. 3MM filter and stained with Coomassie
Brilliant Blue R-250. The proteins were visualized after destaining,
and the filter was dried.
The Damage-independent UvrBC Endonuclease Activity Is Responsible
for the Extra 5' Incision on Damaged DNA Substrates--
Incubation of
a 50-base pair double-stranded DNA substrate containing a cholesterol
adduct attached to the phosphodiester backbone at position 27 in the
top strand (Fig. 1B) with
UvrABC results in incision at the fifth phosphodiester bond 3' of the lesion and at the eighth phosphodiester bond 5' of the lesion (results
not shown). On the same cholesterol substrate containing an artificial
nick at the 3' incision position (Fig. 1C, substrate S2), the 5' incision (producing a 19-nucleotide DNA
fragment) is also efficiently induced (Fig.
2A, lane 2),
confirming previous data that the 5' incision is independent from the
3' incision (7, 8). In addition, a 12-nucleotide incision product is observed, corresponding to an extra 5' incision at 7 nucleotides from
the first 5' incision position. Such an additional 5' incision has been
reported for a variety of DNA lesions (12-14). UvrB*, a truncated form
of UvrB lacking the C-terminal domain, was shown to be defective in 3'
incision on a damaged DNA substrate but fully proficient in 5' incision
(8). Incubation of substrate S2 with UvrA, UvrB*, and UvrC again
results in an efficient first 5' incision but the second 5' incision is
extremely low (Fig. 2A, lane 3). With respect to
the UvrB protein, apparently the requirements for the first and second
5' incision events are different. To study the additional 5' incision
reaction independently from the first 5' incision, substrate S3 was
constructed having an artificial nick at the position of the first 5'
incision site (Fig. 1C). When substrate S3 is incubated with
UvrABC, the 12-nucleotide incision product is observed, indicating that
the first 5' incision event itself is not needed for induction of the
second 5' incision (Fig. 2B, lane 2). Again only
a trace amount of incision (<1%) is observed when wild-type UvrB is
replaced by UvrB* (Fig. 2B, lane 4). No incision
is detected with UvrC or UvrB* alone (results not shown). When
substrate S3 is incubated with only UvrB and UvrC, the 12-nucleotide
incision product is also formed and with an even higher efficiency
(30%) than in the presence of UvrA (13%; Fig. 2B,
lanes 2 and 3). This means that the second 5'
incision is induced by a UvrA-independent UvrBC-associated nuclease.
Also the UvrB*C complex induces residual amount of incision in the absence of UvrA (Fig. 2B, lane 5).
A UvrA-independent UvrBC endonuclease activity has also been
demonstrated on a specific Y DNA substrate (16), in this paper referred
to as substrate S1 (Fig. 1, A and C). We
constructed a similar Y substrate, and incubation of this substrate
with UvrBC results in two incision products (Fig.
3, lane 2). Subsequent analysis on a high resolution gel (results not shown) reveals that the
upper band consists of two fragments of 17 and 18 nucleotides corresponding to incisions events at the third and second
phosphodiester bonds 5' to the single strand-double strand junction,
respectively, which is comparable with the results already described
(16), although the incision frequency in our assay seems much higher. The lower band also consists of two fragments of 10 and 11 nucleotides representing a second 5' incision event 7 nucleotides 5' to the first
incision sites. The positions of these second incision events are
similar to the position of the additional incision in substrates S2 and
S3. The additional 5' incisions on the Y substrate were not reported by
Zou et al. (16), but because in their assay the frequency of
the first incision was much lower, the second incision was probably
below the level of detection. When the S1 substrate is incubated with
UvrB* and UvrC, no incision products are observed (Fig. 3, lane
3), indicating that the UvrC binding domain of UvrB is essential
for the structure-specific incision by UvrBC. Likewise a UvrC mutant
carrying two substitutions (L221P,F223L), which were shown to abolish
the interaction of UvrC with the C-terminal part of UvrB (9), is
equally disturbed in incision of the S1 substrate (results not shown).
This demonstrates that the structure-specific incision of the Y
substrate requires the same protein-protein interaction between UvrB
and UvrC as was found for the extra 5' incision event on damaged
DNA.
To further investigate the similarities of UvrBC-induced incision on
the Y substrate (S1) and the UvrBC-induced extra 5' incision on damaged
substrates, we assayed substrate S3 in the presence of ATP or ADP (Fig.
2B). It has been shown before (16) that both ATP and ADP can
facilitate the UvrBC incision of the Y substrate, albeit ADP with lower
efficiency (77% relative to ATP). Incision of substrate S2 is UvrA-
and ATP-dependent (results not shown), which is expected
because the first 5' incision requires the UvrA- and
ATP-dependent formation of the preincision complex.
Comparable with the Y substrate, incision of substrate S3 is
UvrA-independent and also occurs in the presence of ADP (Fig.
2B, compare lanes 2 and 3 with
6 and 7). With S3 also the efficiency in the
presence of ADP is somewhat lower (24%) compared with that with ATP
(30%). Taken together these results strongly indicate that the UvrBC incision reaction on a Y substrate without damage and the extra 5'
incision reaction on a damaged substrate are identical.
Substrate Requirements for UvrBC Incision--
To test whether the
presence of a DNA lesion in S3 is essential for the extra 5' incision,
a similar undamaged substrate was constructed (Fig. 1C,
substrate S4). This substrate is incised by the UvrBC
endonuclease in the presence of ATP (43%) and ADP (40%) (Fig.
4A, lanes 5 and
6), which is even more efficient than with the damaged DNA
substrate S3 (30 and 24%, respectively). Apparently the DNA lesion is
inhibiting rather than stimulating the incision activity of the UvrBC
nuclease.
We also constructed substrate S5, which lacks the 3' top strand 31 mer
(Fig. 1C). As shown in Fig. 4A (lanes
2 and 3), incision of this substrate is very efficient
(65% in the presence of ATP and 60% in the presence of ADP) and
significantly higher than either of the nicked substrates. This shows
that DNA containing a single strand-double strand junction is better
recognized by the UvrBC endonuclease than nicked DNA; therefore,
substrate S5 was used in all further experiments.
Recently a damage-independent incision on DNA substrates containing a
single-stranded-double-stranded junction also has been reported (15).
The substrates used in these experiments were short oligonucleotides
(25 or 26 nucleotides long) annealed to single-stranded M13 DNA
circles. Comparable with our damage-independent UvrBC incision
activity, the incision in these substrates took place at 7 nucleotides
from the 3' terminus of the oligonucleotide. In contrast to our
results, however, the incision was dependent on UvrA and occurred with
a much lower efficiency (1-5%). The assay reported by Gordienko and
Rupp (15) differs from ours in the presence of a relatively large
amount of single-stranded DNA (M13) and different protein and salt
concentrations. Addition of a comparable amount of M13 single-stranded
DNA (8 fmol) to our incubation mixture indeed results in a severe drop
in the UvrBC nuclease activity (Fig. 4B, compare lanes
1 and 3). This inhibitory effect of single-stranded DNA
seems to be caused by the sequestering of UvrC protein from the
incision reaction, because the use of a higher concentration of UvrC
partly restores the UvrBC nuclease activity (Fig. 4B,
lane 4). With our incubation conditions, however, no
stimulatory effect of UvrA, either in the presence (Fig. 4B,
lanes 4 and 5) or absence (Fig. 4B,
lanes 1, 2, and 6) of M13 single-stranded DNA,
can be detected. The presence of UvrA rather seems to inhibit incision
(Fig. 4B, compare lanes 1 and 5).
Using the incubation conditions described by Gordienko and Rupp (15)
(i.e. 150 mM NaCl instead of 85 mM
KCl), only a very low incision is obtained (Fig. 4B,
lane 7), and indeed with this high salt concentration the
incision becomes UvrA-dependent (Fig. 4B,
lane 8). Because both UvrC and UvrA are single-stranded DNA-binding proteins, it is possible that with the conditions used,
UvrA prevents the sequestering effect of the single-stranded DNA by
competing with UvrC for its binding. In any case, our experiments clearly show that UvrA is not directly involved in the
damage-independent incision reaction.
Protein Domains Needed for UvrBC Incision--
The extra 5'
incision on the 5'-nicked cholesterol-containing substrate S3 is
dependent on the presence of the UvrC binding domain in the C-terminal
part of UvrB (Fig. 2B, lanes 4 and 5). Likewise the UvrBC incision on substrate S5 is disturbed when UvrB*
lacking this domain is used (Fig. 5,
lane 5). With a mutant of UvrC carrying two substitutions in
the corresponding UvrB binding domain (L221P,F223L), a severely reduced
damage-independent UvrBC incision is also obtained (Fig. 5,
lane 7). Apparently, the interaction between the homologous
domains of UvrB and UvrC, which is normally needed exclusively for the
3' incision in damaged DNA substrates, is also involved in the
damage-independent UvrBC nuclease activity. To determine whether UvrBC
uses the catalytic site for 3' or 5' incision for the
damage-independent incision, we also tested the activity of UvrC
(D466A). UvrC (D466A) has a substitution in the catalytic site for the
5' incision reaction, because incubation of damaged DNA substrates with
UvrABC (D466A) results in a normal 3' incision but no detectable 5'
incision (11). Incubation of substrate S5 with UvrBC (D466A) also does
not show any incision activity (Fig. 5, lane 3). As a
control the same UvrC mutant was assayed on the double-stranded
cholesterol-containing fragment in the presence of UvrA and UvrB, and
indeed only the 3' incision product was produced (results not shown).
This means that the damage-independent UvrBC incision activity uses the
catalytic site of UvrC for 5' incision, but in addition it requires an
interaction between UvrB and UvrC, which normally has no function in 5'
incision.
The C-terminal domain of UvrC has also been found to be specifically
involved in 5' incision on damaged substrates. This domain, which is
homologous to the C-terminal domain of the human ERCC1 protein,
contains a helix-hairpin-helix DNA binding motif. A truncated UvrC
protein lacking this C-terminal domain is disturbed in DNA binding and
5' incision. It has been postulated that this motif is needed to
position the catalytic site for the 5' incision reaction (10). As shown
in Fig. 5, lane 9, UvrC554 is also not able to incise
substrate S5, suggesting that the function of the C-terminal domain in
the damage-independent incision is the same as for the 5' incision.
Formation of the UvrBC Protein-DNA Complex--
It has been shown
that incubation of a Y substrate with UvrB and UvrC results in the
formation of a distinct protein-DNA complex in a bandshift gel (16). In
these studies the presence of UvrB in the complex was confirmed, but
the presence of the UvrC protein could not be determined. We assayed
UvrBC·DNA complex formation on substrate S5 and used UvrB and UvrC
antibodies to identify the proteins in the complex. In Fig.
6 a protein-DNA complex is shown, which
is formed in the presence of both UvrB and UvrC (lane 4) but
not with either protein alone (lanes 2 and 3).
Although there is some reaction of the DNA with the preserum
(lane 5), the protein-DNA complex is clearly specifically
retarded in the gel when either antibodies against UvrB (lane
6) or antibodies against UvrC (lane 7) are added,
showing that indeed both proteins are present in the complex. The UvrB*
protein and the UvrC (L221P,F223L) mutant do not give rise to the
UvrBC·DNA complex under the same bandshift conditions (Fig.
7A, lanes 7 and
8), showing the importance of these UvrB-UvrC protein
interaction domains. In contrast, the active site mutant of UvrC
(D466A) does still form the complex (Fig. 7A, lane
9), indicating that its defect in UvrBC nuclease activity is
indeed attributable to the inability to catalyze the incision reaction
itself and not to a defect in substrate binding. UvrC554 is also able
to form the protein-DNA complex (Fig. 7A, lane
4), but the "smearing" of the complex band indicates the UvrBC554 binding is somewhat less stable then in the wild-type complex.
This means that if the helix-hairpin-helix motif of UvrC binds DNA in
the complex, this interaction only marginally contributes to the
stability of the complex, but it seems absolutely essential for the
incision event.
The specificity of DNA binding was tested further using substrate S6
(Fig. 1C). This DNA substrate contains the same lengths of
single- and double-stranded DNA as substrate S5, but the single strand
overhang is on the 3' side. No UvrBC-induced incision can be found
using this substrate, neither in the presence nor in the absence of ADP
or ATP (results not shown), and in accordance, on the bandshift gel no
specific protein-DNA complex is detectable (Fig. 6, lane
9). Apparently UvrBC is not merely binding to the single- or
double-stranded part of the DNA substrate, but it seems capable of
specifically recognizing the 3' end of the single strand-double strand junction.
Interaction between UvrB and UvrC in the Absence of DNA--
From
the bandshift assay described above it is clear that in the UvrBC·DNA
complex the UvrB and UvrC proteins make contact via their homologous
domains. To find out whether this protein-protein interaction is
induced by the DNA or whether it also occurs in solution, a
"bandshift" experiment was done without addition of the DNA
substrate in the reaction mixtures and after electrophoresis the
protein was stained with Coomassie Brilliant Blue.
When UvrC alone is loaded on the gel, the protein becomes visible as a
Coomassie Brilliant Blue-stained band in the slot, indicating that UvrC
is not able to migrate into the gel (Fig. 8, lane 2). UvrC has a
calculated pI of 9.88; therefore, in this gel system (pH 8.5) it is not
expected to be negatively charged. UvrB (pI 4.99) does migrate into the
gel (Fig. 8, lane 1). When both UvrB and UvrC are present,
the UvrB no longer enters the gel, indicating that it is retained in
the slot by UvrC in a UvrB·UvrC complex (Fig. 8, lane 3).
That this retention is indeed indicative for specific complex formation
could be shown by repeating the experiment with UvrB*. The UvrB*
protein migrates into the gel both in the absence (Fig. 8, lane
4) and in the presence of UvrC (Fig. 8, lane 5).
Apparently, also in the absence of DNA a UvrBC complex is formed, via
interaction of the C-terminal domain of UvrB and the homologous domain
of UvrC.
Role of the Nucleotide Cofactor in UvrBC Incision--
The
damage-independent UvrBC incision can take place only in the presence
of ATP or ADP but not without a cofactor (Fig. 4A). Likewise
it has been shown that incision of a Y substrate can occur in the
presence of ATP or ADP but not with a nonhydrolysable form of ATP (16).
Apparently the ADP-bound form of the UvrBC complex is the active one
for incision.
Kinetic analysis of the incision on substrate S5 shows that incision is
very fast: in the presence of ATP 40% incision is reached within 2 min
at 37 °C (Fig. 9A,
lane 2). Incision also takes place at room temperature,
which is as efficient as obtained at 37 °C (Fig. 9A,
compare lanes 10 and 5). During the normal nucleotide excision repair reaction ATP hydrolysis of UvrB is induced
by UvrA and damaged DNA, but apparently the hydrolysis can also be very
efficiently induced by UvrC and nondamaged DNA, even at room
temperature. In the presence of ADP incision is much slower, 10% after
2 min, and after 30 min the incision reaches a level that is slightly
reduced (45%; Fig. 9A, lane 9) compared with
that found with ATP (67%; Fig. 9A, lane 5).
After 60 min of incubation no difference in incision is observed
anymore (Fig. 4A, lanes 2 and 3). This
difference in kinetics probably reflects a lower binding affinity of
UvrB for ADP compared with ATP.
In the bandshift assay as described in Fig. 6, neither ADP nor ATP was
present in the incubation mixture or in the gel onto which the
complexes were loaded. Because for incision the presence of a cofactor
is essential, we also tested the UvrBC·DNA complex formation using
ATP or ADP in the incubation mixture (but not in the gel or gel
buffer). Substrate S5 was incubated with UvrB, UvrC, and cofactor for 5 min at 37 °C before loading on the bandshift gel. Fig. 7A
shows that in the presence of ATP (lane 2) and also in the
presence of ADP, albeit to a lesser extent (lane 3), the UvrBC·DNA complex dissociates. At the same time a band below the unbound DNA becomes apparent, indicating that incision has taken place.
However, the UvrBC·DNA complexes of the UvrC active site mutant D466A
(Fig. 7A, lanes 10 and 11) and of mutant UvrC554 (Fig. 7A, lanes 5 and 6) also dissociate in the
presence of ATP or ADP, showing that it is not the incision itself that
destabilizes the complex but the binding of the cofactor. The
interaction between UvrB and UvrC in solution occurs both in the
absence of nucleotide cofactor and in the presence of ADP or ATP (Fig.
6 and results not shown), indicating that cofactor binding does not
influence the UvrB-UvrC interaction but, rather, the interaction of the UvrBC complex with the DNA.
Because in the UvrBC·DNA complex ATP is hydrolyzed to ADP, we can not
yet conclude whether it is only the ADP-bound UvrB that causes
destabilization of the UvrBC·DNA complex or also the ATP-bound form.
Two different UvrB ATPase mutants were analyzed for protein complex
formation and UvrBC-induced incision. Mutant G509S, with a substitution
in helicase motif V of UvrB, has been shown to induce ATP hydrolysis at
a reduced level (28% of wild type), and mutant R544H, with a
substitution in helicase motif VI, has been shown to be completely
defective in ATP hydrolysis (18). As a consequence, neither of these
UvrB mutants is capable of forming a preincision complex on
damage-containing double-stranded DNA fragments or of inducing incision
on these same substrates. Both in the absence and in the presence of
nucleotide cofactor the two UvrB mutants can bind to UvrC in solution
(Fig. 8 and results not shown). In the absence of cofactor both UvrB
mutants also form a UvrBC·DNA complex with substrate S5 (Fig.
7B, lanes 4 and 7). In the presence of
ATP the complexes of both ATPase mutants dissociate (Fig.
7B, lanes 5 and 8). Because mutant
R544H is fully deficient in ATPase activity, this means that also the
ATP-bound form of UvrB destabilizes the UvrBC·DNA interaction. In the
presence of ADP the UvrBC·DNA complexes of the ATPase mutants are
stable (Fig. 7B, lanes 6 and 9),
suggesting that the mutant UvrB proteins bind ADP very poorly or not at
all. In accordance with this the two UvrB mutants do not show any UvrBC
incision of substrate S5 in the presence of ADP (Fig. 9B,
lanes 4 and 6), whereas mutant G509S, which still
has residual ATPase activity, does partly incise S5 in the presence of
ATP (Fig. 9B, lane 3). The inability of mutant
R544H to hydrolyze ATP and to catalyze incision (Fig. 9B, lane
5) confirms the observation by Zou et al. (16) that
incision can only take place when ADP and not ATP is bound to UvrB.
In summary, our results indicate that in the absence of nucleotide
cofactor the UvrBC complex, which is already formed in solution, can
form a stable complex with the S5 substrate. Whether the protein-DNA
interactions in this complex are mediated via UvrB, UvrC, or both is
still not clear. The binding of ATP (or ADP) to UvrB induces a
conformational change in the protein complex in such a way that the
interaction with the DNA is less stable. In solution the complex is
expected to still be present, because in the presence of cofactor
incision takes place. After entry of the complex in the bandshift gel,
however, dissociation occurs probably as a consequence of the
propensity of the UvrBC-protein complex not to migrate into the gel.
For incision to occur ATP needs to be hydrolyzed, implicating another
conformational change, which probably positions the active site of UvrC
at the incision site.
In this paper we have shown that in the absence of both DNA damage
and UvrA, UvrBC efficiently incises a double-stranded DNA molecule
containing a 5' single strand overhang (S5) and, with a somewhat lower
efficiency, a double-stranded DNA molecule containing a nick in one
strand (S4). This damage-independent incision appears to be responsible
for the additional 5' cutting that takes place after dual incision of
damaged DNA. The same UvrBC nuclease activity was also shown to be
responsible for the structure-specific incision of a Y structure (Ref.
16 and this paper).
In the absence of cofactor UvrBC forms a stable complex with substrates
S5 and and S4 (this paper and results not shown). Substrate S6, which
differs from S5 in the polarity of the double strand-single strand
junction is not bound by UvrBC at all, suggesting that the presence of
a 3' end at a double strand-single strand junction, even if this
junction is merely the result of a nick, is important for substrate
recognition. The Y substrate, which also forms a stable complex with
UvrBC (Ref. 16 and results not shown), however, does not contain such a
3' end. There are no obvious other common denominators in the three
substrates that can explain the binding specificity of the UvrBC
complex. Possibly in S4 and S5 the 3' end of the junction forms the
initial recognition site, and next, as a consequence of the UvrBC
binding, the DNA might adopt a structure similar to that of the Y
substrate. The Y substrate, already having this structure, would then
"fit" right in the UvrBC complex and might therefore not need the
initial recognition site. Although the requirements for UvrBC incision of substrates S4 and S5 very much resemble those for the incision of
the Y substrate, the position of the incision site is different. With
substrates S4 and S5 incision takes place at 7 nucleotides from the 3'
terminus of the strand, whereas with the Y substrate the incision
positions are located 13 and 14 nucleotides from the 3' end. This
implies that the incision position is not dictated by the terminus of
the strand to be incised but more by the structure of the DNA in the
complex. In the Y substrate incision is in the double-stranded portion
of the molecule at the second and third phosphodiester bonds 5' to the
double strand-single strand junction. If indeed binding of UvrBC to S4
and S5 results in partial unwinding of the two DNA strands, this would
create a double strand-single strand junction that subsequently can be
incised at a similar position as in the Y substrate.
The UvrBC damage-independent incision uses the same catalytic site that
on damaged DNA induces the 5' incision. In addition it requires the
presence of the C-terminal DNA binding domain of UvrC, which has also
been shown to be specifically essential for the 5' incision event on
damaged DNA (10). These common features argue that there might be
structural similarities between the UvrBC·DNA complex, which incises
nondamaged DNA, and the UvrBC·damaged DNA complex, in which the 5'
incision takes place. Incision of the undamaged DNA requires that UvrB
in the complex is associated with ADP. For incision of damaged
supercoiled plasmid DNA, it has been shown that addition of UvrC to
purified UvrB·DNA preincision complexes results in incision of the
DNA only when ATP or ATP An important difference between the incision of undamaged DNA and the
5' incision is the role of the C-terminal domain of UvrB. The presence
of this domain is essential for the damage-independent reaction but not
for the damage-dependent incision event. We favor the
hypothesis that the UvrC binding domain is not required for the
incision of the undamaged DNA itself but for the loading of UvrB, in
complex with UvrC, onto the DNA. In damaged DNA this loading is
achieved by UvrA, for which the UvrC binding domain can be omitted. For
the loading of UvrBC onto undamaged DNA, it seems that UvrB and UvrC
first form a complex in solution for which the interaction between the
C-terminal domain of UvrB and its homologous domain in UvrC is required.
The same interaction between the homologous domains of UvrB and UvrC
has previously been shown to be important for the specific binding of
UvrC to the UvrB·DNA preincision complex and the subsequent 3'
incision (8). Binding of UvrC to a UvrAB·DNA complex could not be
demonstrated. It was postulated that the conformational change in UvrB
that accompanies the formation of the preincision complex exposes the
C-terminal domain of UvrB for UvrC to bind to (18). It is now clear,
however, that UvrC can also bind to UvrB when it is not in the
preincision complex. The inability of UvrC to bind to a UvrAB·DNA
complex is therefore more likely the result of shielding the UvrC
binding domain of UvrB by UvrA. In agreement with this, it has been
found that a maltose-binding protein fusion with the last 126 amino
acids of UvrB binds not only to UvrC but also to UvrA (20). If indeed
the binding of UvrB to either UvrA or UvrC is mutually exclusive, this
explains why in our assays UvrA inhibits the UvrBC nuclease activity.
In the presence of ADP or ATP the UvrBC·DNA complex is less stable,
and as a result it can no longer be detected in a gel retardation
assay. This does not necessarily mean that the complex no longer exists
in solution. A Coomassie Brilliant Blue-stained gel shows that in the
absence of DNA the UvrBC complex is retained in the slot, probably as a
result of a lack of negative charge of the UvrC protein at the pH of
the gel buffer. Therefore, only when firmly bound to the DNA can the
complex profit from the negative charge of the DNA to enter the gel.
When this interaction is much weaker, as a result of a conformational
change induced by the nucleotide cofactor, the DNA is expected to be
stripped from the complex during electrophoresis. Destabilization not
only occurs in the ADP-bound complex but also in the ATP-bound form, as
was shown with ATPase mutants of UvrB. Because only the ADP-bound complex seems incision-proficient, this implies that ATP hydrolysis induces yet another conformational change, which probably positions the
catalytic residues at the incision site.
What, if any, might be the in vivo function of the observed
damage-independent UvrBC incision? Because it is clear that UvrA inhibits the UvrBC incision by competing with UvrC for UvrB binding, an
in vivo function can only be expected if in the cell there is an excess of UvrB molecules with respect to UvrA dimers. Several conflicting reports on the amount of Uvr proteins in the cell have
appeared. In unirradiated cells the amounts of molecules per cell have
been reported to be 20 molecules of UvrA and 150-200 molecules of UvrB
(22), 1000 molecules of UvrA and 250 molecules of UvrB (23), and 200 molecules of UvrA and 400 molecules of UvrB (24). In induced cells the
determined quantities were 2200 molecules of UvrA and 1500 molecules of
UvrB (23) and 1200 molecules of UvrA and 2000 molecules of UvrB (24).
If the values as determined by Crowley and Hanawalt (24) are correct,
it should be possible to form UvrBC complexes in the cell, both under
induced and noninduced conditions.
In this paper we have shown that the damage-independent UvrBC nuclease
is responsible for the induction of additional 5' incisions on damaged
substrates. It has been postulated that the function of these
additional incisions is to generate a gap upstream from the DNA lesion,
which can subsequently be used as entry site for RecA-mediated
recombination repair (15). This could in particular be important for
the repair of interstrand cross-links and of closely opposed lesions. A
possible function of the UvrBC nuclease in processes other than DNA
repair, however, should also be considered. Interesting in this respect
is the observation already made a long time ago that the combination of
a mutation in the uvrB gene and a mutation in the
polA gene is lethal to the cell (25), suggesting a possible
role of UvrB in DNA replication. Whether this role is associated with
the UvrBC nuclease activity described in this paper awaits the analysis
of the viability of a *
This work was supported by the J. A. Cohen Institute for
Radiopathology and Radiation Protection (Project 4-2-16).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Characterization of the Escherichia coli
Damage-independent UvrBC Endonuclease Activity*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP
(7000 Ci/mmol, ICN). After incubation at 37 °C for 45 min, the
reaction was terminated at 90 °C for 10 min. The labeled top strand
was hybridized to the bottom strand (4 pmol), and additional top strand
oligonucleotides (4 pmol each) when indicated in the presence of 50 mM NaCl. The substrate was purified by G-50 gel filtration
from the nonincorporated nucleotides in 50 mM Tris-HCl (pH
8.0) and 50 mM NaCl.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (18K):
[in a new window]
Fig. 1.
The 50-base pair DNA substrates used in this
study. A, DNA sequence of substrate S1. The
asterisk indicates the 5'-terminal 32P label.
The arrows indicate the incision positions. The
underlined 11 nucleotides of the bottom strand
are noncomplementary with the top strand. B, DNA
sequence of substrates S2-S6. The structure of the cholesterol lesion
is shown. The cholesterol (Chol) modification is introduced
at position 27 (X) in the top strand. The
arrows indicate the incision positions. The
asterisk indicates the 5'-terminal 32P label.
C, schematic representation of DNA substrates S1-S6. The
numbers indicate the lengths of the different top
strand oligonucleotides. The cholesterol lesion in S2
and S3 is represented by a filled triangle.
View larger version (28K):
[in a new window]
Fig. 2.
Incision of substrates S2 and S3 by
Uvr(A)BC. The 5' end-labeled DNA substrates were incubated with
the indicated proteins at concentrations of 2.5 nM (UvrA),
100 nM (UvrB or UvrB*), and 50 nM (UvrC) for 60 min at 37 °C in Uvr-endo buffer with 1 mM ADP or 1 mM ATP. The incision products were analyzed on a 15%
denaturing acrylamide gel. The incision products of 19 and 12 nucleotides are indicated. A, incision of substrate S2.
B, incision of substrate S3.
View larger version (35K):
[in a new window]
Fig. 3.
Incision of substrate S1 by UvrBC. The
5' end-labeled S1 substrate was incubated with 100 nM UvrB
(lane 2) or UvrB* (lane 3) and 50 nM
UvrC for 30 min at 37 °C in Uvr-endo buffer and 1 mM
ATP. The incision products were analyzed on a 15% denaturing
acrylamide gel. The uncut oligonucleotide (31) and the
products from the first incision (17/18) and the second
incision (10/11) are indicated.
View larger version (41K):
[in a new window]
Fig. 4.
Incision of substrates S4 and S5 by
Uvr(A)BC. The substrates were 5' end-labeled in the top
strand. A, substrates S4 (lanes 4-6) and S5
(lanes 1-3) were incubated with 100 nM UvrB and
50 nM UvrC for 60 min at 37 °C in Uvr-endo buffer with 1 mM ATP or ADP as indicated. B, substrate S5 was
incubated with the indicated amounts (nM) of UvrA, UvrB,
and UvrC for 60 min at 37 °C in Uvr-endo buffer with 85 mM KCl and 1 mM ATP (lanes 1-6) or
in Uvr-endo buffer with 150 mM NaCl and 2 mM
ATP (lanes 7 and 8) in the presence or absence of
8 fmol of single-strand M13 DNA.
View larger version (34K):
[in a new window]
Fig. 5.
Incision of substrate S5 with (mutant) UvrBC
proteins. The 5' end-labeled S5 substrate was incubated with 100 nM UvrB (lanes 1, 3, 7, and 9) or
UvrB* (lanes 4 and 5) and 50 nM UvrC
(lanes 1 and 5) or UvrC (D466A) (lanes
2 and 3) or UvrC (L221P,F223L) (lanes 6 and
7) or UvrC554 (lanes 8 and 9) for 60 min at 37 °C in Uvr-endo buffer with 1 mM ATP.
wt, wild type.
View larger version (53K):
[in a new window]
Fig. 6.
Complex formation of UvrBC with substrates S5
and S6. Substrate S5 (lanes 1-7) and substrate S6
(lanes 8 and 9) were incubated with or without
100 nM UvrB and 50 nM UvrC as indicated for 5 min at 37 °C in Uvr-endo buffer without nucleotide cofactor. Next, 1 µl of preserum (lane 5), anti-UvrB serum (lane
6), anti-UvrC serum (lane 7) was added, and the mixture
was loaded on a 3.5% polyacrylamide native gel.
View larger version (52K):
[in a new window]
Fig. 7.
Complex formation of (mutant) UvrBC with
substrate S5 in the presence or absence of nucleotide cofactor.
A, substrate S5 was incubated with 100 nM UvrB
(lanes 1-6, and 8-11) or UvrB* (lane
7) and 50 nM UvrC (lanes 1-3 and
7), UvrC554 (lanes 4-6), UvrC (L221P,F223L)
(lane 8), or UvrC (D466A) (lanes 9-11), and 1 mM ADP or ATP as indicated. B, substrate S5 was
incubated with 100 nM UvrB (lanes 1-3), UvrB
(G509S) (lanes 4-6), or UvrB (R544H) (lanes
7-9) and 50 nM UvrC (lanes 1-9) with 1 mM ADP or ATP as indicated. The complexes were analyzed on
a 3.5% polyacrylamide native gel. wt, wild type.
View larger version (88K):
[in a new window]
Fig. 8.
Analysis of UvrBC complex formation. 2 µM UvrB (lanes 1 and 3), UvrB*
(lanes 4 and 5), or UvrB (G509S) (lanes 6 and 7) was incubated with (lanes 2, 3, 5, and 7) or without (lanes 1, 4, and 6)
2 µM UvrC for 6 min at 37 °C in Uvr-endo buffer with
100 mM KCl. The incubation mixtures were loaded on a 3.5%
polyacrylamide native gel. After the run the gel was bound to a Whatman
No. 3MM filter, and the proteins were stained with Coomassie Brilliant
Blue R-250. wt, wild type.
View larger version (55K):
[in a new window]
Fig. 9.
Incision of substrate S5 by (mutant) UvrBC
nuclease. A, substrate S5 was incubated with 100 nM UvrB and 50 nM UvrC in Uvr-endo buffer with
1 mM ATP (lanes 1-5, and 10) or 1 mM ADP (lanes 6-9) at 37 °C (lanes
1-9) or at room temperature (lane 10). After
incubation during the indicated times the incision products were
analyzed on a 15% denaturing polyacrylamide gel. B,
substrate S5 was incubated with 100 nM UvrB (lanes
1 and 2), UvrB (G509S) (lanes 3 and
4), or UvrB (R544H) (lanes 5 and 6)
and 50 nM UvrC for 60 min at 37 °C in Uvr-endo buffer
with 1 mM ATP (lanes 1, 3, and 5) or
1 mM ADP (lanes 2, 4, and 6).
wt, wild type.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S is present in the incubation mixture but
not with ADP (21). This would suggest that incision of damaged DNA
requires the ATP-bound form of UvrB. In these studies, however,
incision was monitored by the conversion of supercoiled to relaxed DNA.
Because the induction of the 3' incision alone would also generate
relaxed DNA, it can therefore only be concluded that the 3' incision
event requires ATP. It is very well possible that after the 3' incision
this ATP is hydrolyzed and that subsequently the 5' incision is induced by the ADP-bound form of UvrB, as for the UvrBC cutting of undamaged DNA.
uvrC, polA double mutant.
FOOTNOTES
To whom correspondence should be addressed: Laboratory of
Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, P. O. Box 9502, 2300 RA Leiden,
The Netherlands. Tel.: 31-71-527-4773; Fax: 31-71-527-4537; E-mail:
N.Goosen{at}chem.Leidenuniv.nl.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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