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INTRODUCTION |
The initial events of nucleotide excision repair in
Escherichia coli are controlled by three uvr gene
products: UvrA, UvrB, and UvrC proteins. Our current understanding
suggests that these three proteins work in a sequential manner, with
UvrA first locating and dimerizing at a damaged base on double-stranded
DNA (ds-DNA),1 and UvrB then
binds and forms a Uvr(A)2B complex from which UvrA may
dissociate. Finally, UvrC binds to the Uvr(A)2B complex and triggers dual incisions 6-8 base pairs (bp) 5' and 3-5 bp 3' to the
damaged base. A diverse array of chemically and photochemically induced
modifications to bases in DNA are recognized, including helix-stabilizing and -destabilizing damage (for review, see Refs. 1-3).
Recent studies show that UvrC is essential for 5' and 3' incisions and
is specifically implicated in making the 5' incision through
site-directed mutagenesis studies. UvrC is probably essential for the
catalysis of the 3' incision as well, although it may catalyze this
step itself (4-11).
uvrC differs from uvrA and uvrB in its
genomic organization. It may not be under lexA control as
uvrA and uvrB are (7). The genomic region of
E. coli containing the uvrC gene is complex and
contains at least one overlapping open reading frame (12, 13). The
uvrC gene itself appears to have four promoters and to be
transcribed into two mRNAs (7). The translation start site for the
UvrC protein is also unclear and may be the 5'-GTG upstream of the
first 5'-ATG (13, 14). The precise size of the UvrC protein is
therefore unknown and may be 67,000 Da, or it may be 22 amino acids
longer and 68,500 Da.
The low natural occurrence of the Uvr proteins has necessitated their
study by the use of overproducing cells containing the appropriate
plasmids. This is particularly true for UvrC, because it is estimated
to be present at only <10 molecules per wild-type cell (2).
In this study we have purified UvrC from E. coli strains
containing the overproducing plasmid pDR3274 (15) by several different methods, including a rapid procedure that avoids precipitation of the
protein and expedites isolation of a purified protein. We find that
these preparations yield two different UvrC fractions, which we call
UvrCI and UvrCII according to their elution positions from
single-stranded DNA (ss-DNA) cellulose columns. Both UvrCI and UvrCII
are active in a variety of assays of UvrABC excision nuclease activity
but were found to differ in their interactions with DNA. UvrCII binds
readily to ds-DNA, whereas UvrCI does not. We present evidence that
demonstrates that this UvrCII-DNA binding may affect the UvrABC
excision nuclease activity. These two forms of UvrC were also isolated
from wild-type E. coli cells without containing the pDR3274
plasmids (12). The possible molecular differences between these two
forms of the UvrC protein and their physiological roles are discussed.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes HinfI,
NarI, EoRI, and PstI, T4
polynucleotide kinase, bacterial alkaline phosphatase, acrylamide,
bisacrylamide, agarose, and NACS Prepacs convertible columns
(NACS PACS) were obtained from Life Technologies, Inc. The restriction
enzyme BstNI was obtained from New England Biolabs. Yeast
tRNA was obtained from Sigma. All 32P-labeled nucleotides
were obtained from Amersham Pharmacia Biotech NEN Life Science
Products, Inc. Nitrocellulose membranes (hydrophilic, 0.45 µM, HAWP) were purchased from Millipore.
DNA and DNA Fragment Isolation--
X174 replicative form I
(RFI) DNA and plasmid pBR322 were isolated and purified by cesium
chloride density gradient centrifugation. T7 phages were prepared by a
method described by Yamamoto and Alberts (16). T7 DNAs were prepared by
removing proteins from phages by phenol and diethyl ether extractions.
The 247-bp HinfI-BstNI single 3'
end-32P-labeled fragments of pBR322 were isolated from a
383-bp BstNI fragment that had been agarose gel-purified and
3' end-labeled with [
-32P]dTTP. The 174-bp
EcoRI-HaeIII single 5'
end-32P-labeled fragments of pBR322 were prepared as
described previously (17). The EcoRI-PstI 750-bp
fragments of pBR322 were isolated from agarose gel electrophoresis and
32P-labeled with [
-32P]ATP at both 5' ends.
UV Irradiation--
X174 RFI DNA,
[3H]thymidine-labeled T7 DNA, and 32P-labeled
defined DNA fragments were irradiated with a germicidal lamp (Sylvania, C15T8; major emission, 254 nm) to produce eight dimers or one dimer per
DNA molecule.
Purification of Uvr Proteins--
UvrA, UvrB, and UvrC proteins
were purified from the E. coli K12 strain CH296
(uvrC34)- and DR1984 (recA1 uvrC34)-carrying plasmids pUNC45 (uvrA), pUNC211 (uvrB), or
pDR3274 (uvrC; Ref. 16). These plasmids and E. coli strains were kindly provided by Dr. A. Sancar (University of
North Carolina, Chapel Hill, NC). The pUVC1234 plasmid construct (12),
which contains the endogenous uvrC operon, was a generous
gift from Dr. R. Moses (University of Oregon, Portland, OR). UvrC
protein was also purified from E. coli cells (MST 1) with
the wild-type uvrC gene (17, 18).
UvrABC Excision Nuclease Reactions--
The UvrABC excision
nuclease reaction was conducted in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 1 mM ATP, and 1 mM dithtiothreitol. An aliquot of
32P-labeled DNA or
X174 RFI DNA (0.2 µg) was reacted
with 15 nM UvrA, 15 nM UvrB, and 15 nM UvrC in a volume of 25 µl for 60 min at 37 °C. For
X174 RFI DNA the reactions were stopped by adding 0.1% SDS and
heating at 65 °C for 5 min, and the DNAs were then electrophoresed in a 1% agarose gel in buffer containing 40 mM Tris acetate, pH 8.0, and 1 mM EDTA at 1 V/cm for 14 h. The gel was stained with 0.5 µg/ml ethidium
bromide to visualize the supercoiled and relaxed forms of DNA. The
pictures were scanned by a Bio-Image Analyzer with a Visage 100 whole-band analysis software program. For 32P-labeled DNA
fragments the reactions were terminated by phenol extractions. The
labeled DNA was then ethanol precipitated, washed in 75% ethanol,
dried, and dissolved in a formamide denaturing dye mix (80% v/v
formamide, 0.1% xylene cyanol, and 0.1% bromphenol blue).
Sequencing Gel Electrophoresis--
Chemical sequencing was
carried out as described by Maxam and Gilbert (21) with the
modifications described (17). DNA samples were heated at 90 °C (2 min) and quenched in an ice bath. The samples were applied to a
sequencing gel, consisting of 8% acrylamide and 7 M urea
in Tris borate-EDTA buffer (50 mM Tris-HCl, 50 mM sodium borate, and 10 mM EDTA, pH 8.3), in
parallel with the Maxam and Gilbert (21) sequencing reactions. After
electrophoresis the gel was dried in a Bio-Rad gel dryer and exposed to
Eastman Kodak Co. X-Omat RP films at
70 °C for various lengths of
time. The intensity of bands was determined by scanning with a
Bio-Image Analyzer as described above.
DNA Binding Assay--
32P-labeled pBR322 DNA
fragments or linearized
X174 RFI DNA, and
[3H]thymidine-labeled T7 DNA were incubated with UvrC or
UvrA at different protein/DNA ratios in UvrABC excision nuclease
reaction buffer for 60 min at 37 °C. At the end of incubation the
mixtures were chased with an excessive amount of calf thymus DNA for
10 s, filtered through a membrane (0.45 µM; HAWP,
Millirpore), and washed with UvrABC excision nuclease reaction buffer
without ATP for 10 s or electrophoresed in a 0.5% agarose gel in
buffer containing 50 mM Tris, pH 7.9, 50 mM
acetate, and 1 mM EDTA. The radioactivity in the dried
membranes was counted in an LKB 1219 scintillation counter. The agarose
gels were air-dried and exposed to Kodak X-Omat RP films at
70 °C
for various times. The intensity of bands was determined by scanning
with a Bio-Image Analyzer.
N-terminal Amino Acid Analysis--
To determine the N-terminal
amino acid sequence of UvrC, the proteins were separated by 10%
SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a
polyvinylidene difluoride membrane. The UvrC band was eluted, and the
first 12 N-terminal amino acids were determined by Beckman amino acid
sequences according the method as described by Matsudaira (22).
UvrC Cross-link Reactions--
Aliquots containing 1 µg of
UvrC were incubated with 0.1% glutaraldehyde at room temperature for
10 min. Immediately after the reaction the proteins were denatured by
boiling in SDS solution for 5 min, separated by 7% SDS-PAGE, and then
transferred to a nitrocellulose membrane.
Immunoblotting--
7 or 10% acrylamide, 0.32% bisacrylamide,
and SDS were used as described by Laemmli (23). After electrophoresis
the proteins were electrotransferred to a nitrocellulose membrane and
reacted with polyclonal UvrC antiserum (18); the antigen-antibody
complex was further conjugated with horseradish peroxidase-labeled
antibodies and then detected by chemiluminescence (24).
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RESULTS |
Purification of Two Forms of UvrC by ss-DNA Cellulose
Chromatography--
Several purification schemes have been reported
for UvrC purification, and they have in common two steps:
phosphocellulose and ss-DNA-cellulose chromatography (15, 25, 26). UvrC
proteins can be precipitated with polymin P before going through
chromatographic separation (25). In these procedures UvrC is eluted
last from an ss-DNA-cellulose column with 0.65 M KCl. We
found that substituting a KCl gradient for the more usual step gradient
to elute the ss-DNA-cellulose column results in the elution of two
distinct peaks of A280 material, and both peaks
have UvrC activity (Fig. 1A;
details are described in the following sections).

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Fig. 1.
Separation of two forms of UvrC by
ss-DNA-cellulose chromatography. The UvrC proteins from cell
lysates of CH296/pDR3274 were either precipitated with polymin P
(0.5%; A) or left without this precipitation
(B). Partially purified UvrC collected from combined peak
fractions of phosphocellulose chromatography was diluted to 0.2 M KCl in buffer A and applied to a 1.5 × 14-cm column
containing 0.5-1 g ss-DNA-cellulose. The column was washed and
developed with a linear gradient of KCl (0.3-1 M KCl;
dotted lines) at a flow rate of 0.5 ml/min, and the 4-ml
fractions were collected. A and B, wavelength
280-nm absorbance profile of the eluted fractions (solid
lines). C, SDS-PAGE analysis of proteins in the
different fractions from B. Lane 1, molecular
standards; their molecular masses (in kilodaltons) are indicated at the
left.
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Because the two-peak elution profile of the UvrC protein has never been
reported before, we then explored the possibility of whether the
multistep purification procedure for UvrC was responsible for this
striking pattern by simplifying the purification scheme. UvrC was
purified from a cell sonicate (CH296 or DR1984 cells containing pDR3274
and induced with 0.5 mM isopropyl
-D-thiogalactoside) prepared in 0.3 M
KCl. The supernatant after centrifugation was purified through a
phosphocellulose column and then an ss-DNA-cellulose column without
going through a polymin P precipitation step. Elution of the
ss-DNA-cellulose column with a KCl gradient gives a similar profile as
in Fig. 1A, showing two peaks of protein (Fig.
1B). Both peaks contain a major (>95%) UvrC band of a
molecular mass of 65,000 Da when separated by SDS-PAGE (Fig.
1C); the amount of UvrC collected in the first peak
fractions is approximately twice as much as in the second peak
fractions. We refer to the two fractions of UvrC according to their
elution positions as UvrCI (0.4 M KC1) and UvrCII (0.6 M KC1).
UvrCI and UvrCII Are Active in Incising UV-irradiated, Supercoiled
X174 RFI DNA--
The activity of these two forms of UvrC in
incising UV-induced DNA damage was tested. Various amounts of UvrC
proteins were reacted with UV-irradiated
X174 RFI DNA (2.2 nM) in the presence of UvrA (15 nM) and UvrB
(15 nM). (We term this reaction condition, which has a
relatively high nucleotide/Uvr ratio, condition I). The results in Fig.
2 show that both forms of UvrC were
active in incising UV-irradiated, supercoiled DNA. However, the
specific activity of UvrCII is lower than that of UvrCI; the former is one-fourth as active in incising UV-irradiated
X174 RFI DNA as the
latter.

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Fig. 2.
UvrABCI and UvrABCII nuclease incisions of
UV-irradiated supercoiled DNA. Supercoiled X174 RFI DNA
containing eight pyrimidine dimers per DNA molecule
(U8) was reacted with various amounts of either
UvrCI or UvrCII (1 represents 15 nM; the molar
concentration was calculated on the basis of the assumption that the
molecular mass of both UvrC forms is 65 kDa according to the results
shown in Fig. 1C) in combination with UvrA (15 nM) and UvrB (15 nM) under standard reaction
conditions at 37 °C for 60 min, and the resultant DNA was separated
by electrophoresis in a 1% agarose gel. A, typical gel.
Top panel, individual Uvr proteins used in the incision
assay. U0, unirradiated X174 RFI DNA; +, Uvr
protein presence; , Uvr protein absence; CCC, covalently
closed circle; OC, open circle. B, effect of UvrC
concentration on the incision of U8 DNA. The fraction of
U8 incised was quantified from densitometer scanning of
bands corresponding to CCC and OC in
A.
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Because the majority of proteins present in both peaks have a molecular
mass (denatured by boiling in SDS solution) corresponding to that of
UvrC, their N-terminal amino acid sequences were determined. We have
found that the first 12 of the 13 N-terminal amino acids of both forms
of UvrC are the same; starting from the second amino acid the sequence
is: Asp-Gln-Phe-Asp-Ala-Lys-Ala-Phe-Leu-Lys-Thr-Val.
UvrCII Is a Tetramer, and UvrCI Is a Monomer--
Because the
monopeptides of UvrCI and UvrCII not only have the same molecular mass
but also have the same N-terminal amino acid sequence, and no other
peptides have been found in association with these two forms of
proteins (Fig. 1), the results raise the possibility that these two
forms of UvrC could result from different folding or that the native
form of these two proteins may be composed of different numbers of
monopeptides, or both. To test these possibilities we have determined
the molecular mass of these two forms of UvrC proteins by
glutaraldehyde cross-link reaction and size exclusion chromatography
(27). Fig. 3 shows that treatment of
UvrCI with 0.1% glutaraldehyde resulted in one additional band
corresponding to molecular mass of 130 kDa besides the 65-kDa band. In
contrast, the same glutaraldehyde treatment of UvrCII resulted in two
additional bands; one corresponding to 130 kDs, the same as observed
with UvrCI treatment, and the other having a molecular mass >240 kDa. Results from size exclusion chromatography show that UvrCI eluted at a
major peak corresponding to the bovine albumin position (68 kDa). In
contrast, the UvrCI-UvrCII mixture eluted at two peaks corresponding to
~68 and >232 kDa, respectively (Fig. 3B). Together, these
results suggest that UvrCI is a monomer, and UvrCII is a tetramer.

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Fig. 3.
Determinations of UvrCI and UvrCII molecular
mass by size exclusion chromatography and glutaraldehyde cross-linking
reactions. A, typical gel filtration profile of UvrCI
and UvrCII. UvrCI and UvrCII purified from an ss-DNA-cellulose column
were separated by Sephacryl S-300 HR gel chromatography (eluted with
0.55 M KCl); a, separation of a mixture of UvrCI
and UvrCII; b, UvrCI only; c, UvrCII only. The
positions of the molecular mass standards are indicated by
arrows. B, typical Western blotting profile of
UvrCI and UvrCII after glutaraldehyde treatment. UvrCI and UvrCII
proteins were reacted with glutaraldehyde, denatured as described,
separated by 7% PAGE, and transferred to a nitrocellulose membrane.
The membrane was reacted with UvrC antibodies followed by horseradish
peroxidase antibodies. The antigen-antibody reactions were detected by
the chemiluminescence method (24). Lane 1, UvrCI mock
treated; lane 2, UvrCI treated with 0.1% glutaraldehyde at
room temperature for 10 min; lane 3, UvrCII mock treated;
lane 4, UvrCII treated with glutaraldehyde as in lane
2. The positions of the molecular standards (in kilodaltons) are
indicated.
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UvrCI and UvrCII React to UV-irradiated Linear DNA
Differently--
It has been well established that the UvrABC excision
nuclease makes dual incisions 6-8 bp 5' and 3-5 bp 3' to UV-induced pyrimidine dimers and other chemical-DNA adducts (for review, see Refs.
1-3). However, it has been found that occasionally the 5' and 3'
incisions induced by UvrABC may uncouple, and the uncoupled incision
occurs in UV-, bulky chemical carcinogen-, and CC-1065-modified
DNA (for review, see Ref. 2). Selby and Sancar (28) have reported that
"aged" UvrC proteins may lose their 5' incision ability. To
determine whether UvrCI and UvrCII, in combination with UvrA and UvrB
(we term the collective function of Uvr proteins UvrABCI and UvrABCII
excision nuclease, respectively), would have the same dual incision
activity, UV-irradiated single 3'-end-32P-labeled
HinfI-BstNI 247-bp pBR322 DNA fragments (1 nM) or single 5'-end-32P-labeled
EcoRI-HaeIII 174-bp pBR322 DNA fragments were
reacted with 15 nM of these proteins (we term this reaction
condition, which has a relatively low nucleotide/Uvr ratio, condition
II). The results are shown in Fig. 4;
although UvrABCI makes the expected dual incisions at pyrimidine
dimers, under the same reaction conditions UvrABCII does not incise the
same UV-irradiated linear DNA fragments (Fig. 4, A, compare
lanes 11 and 15, and B, compare
lanes 12 and 17).

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Fig. 4.
A, UvrABCI and UvrABCII incisions of
UV-irradiated (one pyrimidine dimer per DNA fragment; lanes
11-18) and unirradiated (lanes 1-6) 3'
end-32P-labeled 247-bp BstNI-HinfI
pBR322 DNA fragments. The DNA fragments were incubated in the standard
reaction mixture containing 15 nM UvrA, 15 nM
UvrB, and 15 nM UvrCI or UvrCII and with different amounts
of competitive DNA (unirradiated, nonlabeled linear X174 RF ds-DNA
as indicated at the top): lanes 11 and
15, 0 µg; lanes 12 and 16, 0.1 µg;
lanes 13 and 17, 1 µg; lanes 14 and
18, 2 µg. Lanes 1-6, reactions using
unirradiated DNA fragments. The mixtures were incubated at 37 °C for
60 min, and the resultant DNAs were electrophoresed in a sequencing
gel. The Maxam and Gilbert (21) A+G, G, T+C, and C sequencing
reactions are indicated. Uvr protein treatments were as follows:
Lane 1, no enzyme; lane 2, UvrA only; lane
4, UvrCI; lane 3, UvrCII; lanes 5 and
11-14, UvrABCI; lanes 6 and 15-18,
UvrABCII. B, UvrABCI and UvrABCII incisions of
UV-irradi- ated (one pyrimidine dimer per DNA fragment; lanes
12-21) 5' end-32P-labeled 174-bp
EcoRI-HaeIII pBR322 DNA fragments. The conditions
for UvrABC reactions and the sequencing gel electrophoresis are the
same as described in A. The amounts of competitive DNA added
are indicated at the top. The pyrimidine tracts (lanes
1-16), which contain two or more contiguous pyrimidines and which
have the potential to form photodimers, are indicated in the left
panel. The photodimers induced corresponding UvrABC incision bands
(u1-u16) are indicated in the right panel.
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There are two possible explanations for these unexpected results; one
is that UvrABCII is inactive in incising photodimers in linear DNA, and
the other is that UvrCII may interfere with Uvr(A)2B-photodimer complex formation. It has been reported
that the presence of excessive UvrA protein inhibits UvrABC excision activity, and the inhibition has been speculated to result from the
binding of excessive UvrA proteins to damaged bases, which consequently
prevents proper Uvr(A)2B-DNA damage complex formation (29,
30). The inability of UvrABCII to incise the small quantity of
UV-irradiated linear DNA fragments shown in Fig. 4 cannot be attributable to an excessive amount of UvrA proteins, because the
active UvrABCI reaction conditions contain the same amount of UvrA
proteins as in the inactive UvrABCII reaction conditions. One of the
major differences between conditions I and II is that although the
nucleotide/UvrC ratio is 770 in condition I, it is between 10 and 17 in
condition II; the difference between these two conditions is
45-77-fold. If the UvrCII protein is able to bind to ds-DNA and UvrCI
is not, then the formation of a proper Uvr(A)2B-DNA damage
complex may be affected by excessive amounts of UvrCII (but not UvrCI),
similar to the effect of excessive UvrA proteins. To test this
possibility we added different amounts of unirradiated, linearized
X174 RF DNA (competitive DNA) in condition II to reduce the
protein/nucleotide ratio. The results in Fig. 4 show that although
additional DNA, ranging from 0.05 to 0.2 µg, enhances UvrABCI
excision activity, it affects UvrABCII incision activity more
drastically, restoring the incision activity of UvrABCII to a level
comparable with that of UvrABCI. These results also show that UvrABCII,
in the presence of competitive DNA, makes dual incisions 6-8 bp 5' and
3-4 bp 3' of a photodimer in the substrate DNA in the same manner as
UvrABCI does. It is worth noting that in the presence of excessive
competitive DNA the incision activities of both UvrABCI and UvrABCII
are reduced (Fig. 4, A, lanes 14 and 18, and
B, lanes 16 and 21).
UvrCII Is a Double-stranded DNA-binding Protein, and UvrCI Is
Not--
The above results suggest that UvrCII may be a ds-DNA-binding
protein, whereas UvrCI is not. To test this possibility the conventional nitrocellulose filter binding assay for two forms of UvrC
and UvrA proteins was performed. Different amounts of Uvr proteins were
added to a fixed amount of 3H-labeled T7 DNA (7.3 fmol)
with or without UV irradiation to produce 8 dimers per DNA fragment.
The protein-DNA mixtures were incubated in the standard UvrABC reaction
solution for 60 min at 37 °C and then were chased with an excessive
amount of cold DNA before filtering through nitrocellulose membranes.
Results in Fig. 5 show that UvrCII indeed
binds to ds-DNA, whereas UvrCI does not, and UvrCII-DNA binding is
linearly proportional to UvrCII concentrations. Furthermore, it appears
that there is no significant difference between the binding affinities
of UvrCII to UV-irradiated and unirradiated DNA. These two features are
in great contrast to UvrA-DNA binding, which is exponentially
proportional to UvrA concentrations and shows higher affinity toward
UV-irradiated DNA than nonirradiated DNA (31). Consistent with these
filter binding assay results, Fig. 6
shows that the presence of UvrCI proteins does not affect the mobility
of the DNA fragments; in contrast, the presence of UvrCII proteins
retards the mobility of the labeled DNA fragments significantly.
Scatchard plotting (with the assumption that UvrCII is a tetramer with
a molecular mass of 272 kDs on the basis of cross-linking reaction
results) renders the binding constant of UvrCII-ds-DNA of 9 × 108 M
1 (Fig. 6),
which is
-
that of UvrA. It is worth noting that
a significant portion of the retarded DNA fragments distributes in a
smear (in an overly exposed film; data not shown); the smear may result
from dissociation of UvrCII from DNA during electrophoresis.

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Fig. 5.
DNA binding of UvrCI ( and ), UvrCII
( and ) and UvrA ( and ). Different concentrations of
proteins were incubated with a fixed amount of
[3H]thymidine-labeled T7 DNA (7.3 fmol) with
(filled symbols) or without (open symbols) UV
irradiation (to produce eight dimers per DNA molecule) in the standard
UvrABC reaction solution for 60 min at 37 °C. At the end of
incubation an excessive amount of calf thymus DNA was added for 10 s, and the mixture was filtered through a 0.45-µm nitrocellulose
membrane. DNA retained on the membrane was measured and is interpreted
to be protein-bound. All data points are the average of
duplicate assays.
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Fig. 6.
A, gel electrophoresis of DNA fragments
reacted with UvrCI and UvrCII. Aliquots containing
32P-end-labeled EcoRI-PstI 750-bp
pBR322 DNA fragments (14 fmol) were incubated with different amounts of
UvrCI (0, 84, 168, 336, and 672 fmol) or UvrCII (0, 84, 168, 252, 336, 420, 504, and 672 fmol) under the same conditions as described in Fig.
5. At the end of the incubation the protein-DNA mixtures were
immediately electrophoresed in a 0.5% agarose gel in Tris borate-EDTA
buffer. The autoradiographs were scanned, and the binding data were
plotted as shown in B. Data points were obtained
from results of both the gel retardation assay and the filter binding
assay. Inset, Scatchard plot of the binding data (from the
gel retardation assay). The average number of UvrCII bound per DNA
fragment ( ) was calculated from the distribution of DNA in the
various bands as a direct average: = nfn/ fn, where
fn is the fraction of DNA fragments that has
n proteins bound (31). The percentage of DNA bound in the
filter binding assay was normalized by using the highest binding as
100%.
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Relationship between UvrCII-DNA Binding and UvrABCII Incision
Activity--
To determine the effect of UvrCII-DNA binding on
UvrABCII incision activity, 3' end-32P-labeled,
UV-irradiated HinFI-BstNI 247-bp DNA fragments (1 nM) were incubated with 15 nM UvrCII in
standard reaction conditions for 60 min at 37 °C, different amounts
of competitive DNA (nonlabeled linearized
X174 RF DNA) were added,
and the mixtures were further incubated for another 60 min at 37 °C
with or without UvrA and UvrB proteins. At the end of the incubation
the sites and the extent of incision of these DNAs by UvrABCII (Fig.
7) and the percentage of labeled DNA
binding to UvrCII (Fig. 8) were determined by sequencing gel
electrophoresis and filter binding assay, respectively. The incision
activity of UvrABCII increases as the amount of the additional
competitive DNA increases (Fig. 7). Conversely, the results in Fig.
8 show that the fraction of damaged DNA
bound with UvrCII decreases exponentially as a function of the
concentrations of the competitive DNA. The relationship between the
reduction of damaged DNA bound with UvrCII and the increase of UvrABCII incision is better demonstrated in Fig. 8, which shows that the incision reaches a plateau level when UvrCII-damaged DNA binding is
reduced to a few percentiles, and further addition of competitive DNA
slightly reduces incision. These results are consistent with the
interpretation that the UvrCII-DNA binding hinders
Uvr(A)2B-DNA damage complex formation and
consequently reduces UvrABC incision activity.

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Fig. 7.
Effect of unirradiated, competitive DNA on
the incision of UV-irradiated substrate DNA by UvrABCII excision
nuclease. The 3'-end-labeled, UV-irradiated 247-bp
BstNI-HinfI pBR322 DNA fragments (1 nM) were reacted with UvrCII (15 nM) in the
standard UvrABC reaction solution in the presence of different amount
of competitive DNA (as indicated at the top) for 60 min at
37 °C. At the end of the incubation the mixtures were either
filtered through a nitrocellulose membrane as described in Fig. 5 or
further incubated for 60 min after the addition of UvrA (15 nM) and UvrB (15 nM). The resultant DNAs were
separated by electrophoresis in a sequencing gel as described in Fig.
4. The symbol and lane descriptions are the same
as in Fig. 4.
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Fig. 8.
Relationship between UvrCII-DNA binding and
UvrABCII incision of UV-irradiated substrate DNA. The percentage
of 32P-labeled, UV-irradiated DNA fragments retained in the
filter attributable to UvrCII binding was calculated as described in
Fig. 6. The percentage of incision was calculated from the densitometer
scanning results of Fig. 7 (total intensity of u1-u16)
using the highest cutting as 100% (Fig. 7, lane
17).
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Isolation of UvrCI and UvrCII from E. coli Cells with and without
Plasmids Containing the Endogenous uvrC Operon--
Because pDR3274 is
a recombinant plasmid with the uvrC structural gene sequence
linked to the tac promoter (15), it was possible that the
two forms of UvrC proteins produced by this plasmid may be different
from that produced by the native endogenous uvrC operon. To
test this, we isolated UvrC, from E. coli cells without
harboring the pDR3274 plasmid. Cell lysates were chromatographed through phosphocellulose and ss-DNA-cellulose columns following the
procedures described above. Although the wild-type cell lysates did not
render two clear A280 absorbance peaks
corresponding to 0.4 and 0.6-0.7 M KCl, Western blotting
results show that two peaks of UvrC protein were separated and eluted
at these two KCl concentrations (Fig. 9).
Together, these results suggest that two forms of UvrC are produced by
the endogenous uvrC operon.

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Fig. 9.
Purification of two forms of UvrC from
wild-type E. coli cells harboring no uvrC
plasmids. A typical immunoblotting result is shown. The
purification procedures were the same as described in Fig. 1. The
proteins eluted from an ss-DNA-cellulose column by a KCl gradient were
separated by electrophoresis using 12% SDS-PAGE, electroblotted to a
nitrocellulose membrane, and detected using UvrC antibodies as by
described in Fig. 3. UvrC proteins were eluted at fractions at ~0.4
M KCl and at fractions between 0.6 and 0.7 M
KCl with no protein detected in the intermediate salt range.
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DISCUSSION |
The functions of uvrC are the least understood among
the three uvr genes, uvrA, uvrB, and
uvrC, which are involved in the initial incision step of
nucleotide excision repair. Although in vitro it has been
demonstrated that the addition of UvrC proteins to a
Uvr(A)2B-DNA damage complex induces dual incisions 5' and 3' to damaged bases, several laboratories have reported that UV irradiation induces DNA single-strand breaks in uvrC34
mutant cells but not in uvrA and uvrB mutant
cells, even though these three uvr mutant cells are unable
to excise cyclobutane pyrimidine dimers (19, 32-34). Using a viral
E. coli transfection system we have found that only
uvrC mutant cells show low transfectivity for viral DNA
containing N-(guanosin-8-yl)-2- aminofluorene adducts; uvrA and uvrB cells show the same transfectivity
as do wild-type cells. In contrast, these three mutant cells have the
same low transfectivity for UV-irradiated or
N-acetoxy-2-acetylaminofluorene and anti- and
syn-benzo(a)pyrene diol epoxide-modified viral DNA (20, 35).
These results indicate that uvrC gene products may function
in a manner more complicated than simply participating in the incision
step and may interact with proteins other than UvrA and UvrB in
vivo.
The understanding of UvrC function has been hampered by the scarcity of
these proteins in cells and their instability. Purification of UvrC
proteins has been achieved by using
lysogens with the uvrC gene (36, 37) or cells with a plasmid containing the uvrC gene (12). The method of purification is mainly based
on the ability of UvrC proteins bind to ss-DNA. Yeung et al.
(26) have eluted purified UvrC protein at 0.45 M KCl;
however, using a step gradient, Thomas et al. (15) have
reported that UvrC proteins were eluted completely at 0.65 M KCl. Our modified method further separated the UvrC into
two peaks, one at 0.4 M KCl and another at 0.6 M KCl. It appears that the quantity of ss-DNA-cellulose used for column chromatography and the elution rate are the two critical factors for separating these two forms of UvrC proteins; we
observed a single UvrC peak, which contained both UvrCI and UvrCII
activities when the purification procedure involved a small quantity of
ss-DNA-cellulose and a fast elution rate (data not shown).
We have found that these two forms of UvrC proteins can be further
purified by ds-DNA-cellulose chromatography but with low recovery
(20%); the reason for this low recovery is not clear. However, we also
have found that the two forms of UvrC prepared by ss-DNA-cellulose
chromatography and those further purified by ds-DNA-cellulose
chromatography have the same mobility in SDS-PAGE and the same pI
(native form, 7.48; denatured form, 9.4), and in the presence of UvrA
and UvrB, both showed a dual incision pattern on UV-irradiated DNA. The
sources causing the differences in the ds-DNA binding and elution
pattern in ss-DNA chromatography are unknown. Contrary to published
results, we have found that the N termini of these two forms of UvrC
proteins we have purified are not blocked, and the first 12 of the 13 N-terminal amino acids, starting from the second amino acid, are
consistent with the result reported by Moolenaar et al. (13)
that the initiation codon for the uvrC gene starts at the
5'-GTG of positions 882-884 rather than at the 5'-ATG of positions
772-774 (14). The source of this discrepancy is unclear.
Because the peptide in the two forms of UvrC have the same molecular
mass and N-terminal amino acid sequence, it is possible that UvrCII may
have resulted from aggregation of UvrCI. Although we are unable to
exclude this possibility entirely, several lines of evidence suggest
this may not be the case. First, so far we are unable to convert either
form of UvrC to the other by treatments such as oxidation and
reduction. Second, we show that only monomeric and tetrameric, but no
dimeric and trimeric, UvrC proteins are obtained by size exclusion
chromatography. Third, in the electrophoretically separated products of
UvrCII treated with the cross-linking reagent glutaraldehyde, we
observed mainly the monomeric and tetrameric, but no trimeric, forms of
UvrC. Furthermore, we observed the existence of these two forms of UvrC
in wild-type cells, even though the copy number of the UvrC proteins in
these cells is very low (~10 copies per cell).
It is possible that the difference in ds-DNA binding resides in
post-translation modification or in the association of cofactors, or
both. If one UvrC form is modified or associated with some cofactors
and the other is not, then the two UvrC proteins most likely would be
observed in overproducing cells for the simple reason that overproduced
UvrC proteins oversaturate the post-translation modification capacity
or the amount of cofactors present in the cells. However, using the
same purification protocol, we have isolated UvrC forms that eluted at
0.4 M KCl as well as at 0.6-0.7 M KCl from
wild-type E. coli cells harboring no recombinant
uvrC plasmid and E. coli cells with pUVC1234
plasmids containing the endogenous uvrC operon (12). Because
of the minute quantity of UvrC purified from these cells, we were
unable to determine their activities. However, because even in cells
producing minute amounts of UvrC proteins we were able to isolate UvrC
that had an elution profile identical to that of UvrCI and UvrCII, it
is likely that these two forms of UvrC proteins are the normal
uvrC gene products and are not the results of overproduction.
We speculate that UvrCII is a major form existing in wild-type cells
for two reasons. One is that UvrCII should be active in incision
in vivo, because the cellular nucleotide/protein molar ratio
is relatively large. The second reason is that because each cell has
only <10 molecules of UvrC, and UvrCII does not form a complex with
the free forms of the UvrA or UvrB protein, to account for the fast and
efficient excision repair in vivo, the UvrC proteins must be
concentrated in the genome area. The loose association of UvrCII to DNA
would fulfill this critical requirement.