From the Department of Environmental and Molecular
Toxicology, Oregon State University,
Corvallis, Oregon 97331-7301, the § Department of
Biophysics and Biochemistry, University of Rochester Medical Center,
Rochester, New York 14642-8408, and the ¶ Department of
Pathology, University of Kentucky Medical Center,
Lexington, Kentucky 40536
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ABSTRACT |
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Previous studies have demonstrated recognition of
DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee,
E., and Hays, J. B. (1991) Genetics 129, 1007-1020)
and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed
specifically against incorrect bases inserted during semi-conservative
DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T
cis-syn-cyclobutane pyrimidine-dimers or T-T
pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible
bases opposite the respective 3'-thymines in the photoproducts, were
analyzed for the ability to compete with radiolabeled (T/G)-mismatched
DNA for binding by highly purified human MSH2·MSH6 heterodimer
protein (hMutS In most prokaryotes, and in all eukaryotes examined, highly
conserved protein systems that recognize DNA mismatches and certain DNA
lesions play critical roles in maintenance of genetic stability. These
long-patch mismatch-repair systems decrease DNA replication error rates
100 The MutS/MutL recognition/coupling paradigm is retained in eukaryotes
but is now more complex (4, 5). The single eubacterial MutS is replaced
by at least six MutS homologs
(MSHs)1 in yeast, human
beings, and probably other eukaryotes. Recognition of nuclear-DNA
mismatches is accomplished by two distinct heterodimers, comprised of
MSH2 plus MSH6 or MSH2 plus MSH3 polypeptides; these show different but
overlapping specificities for base/base mismatches and various
insertion/deletion loop-outs (6-9). Other MutS homologs, MSH1, and
MSH4 and MSH5, play less well characterized roles in mitochondrial
stability (10) and meiotic recombination (11, 12) respectively. Two of
the several eukaryotic MutL homologs, in the form of an MLH1·PMS2
heterodimer, couple mismatch recognition to subsequent processing steps
(13); this again appears to involve ATP-triggered translocation of
MSH2·MSH6 (or MSH2·MSH3) (14, 15).
Accumulating evidence for interaction of mismatch-repair systems with a
surprising variety of DNA lesions now points to additional roles for
mismatch repair. These lesions include UV light photoproducts (16, 17),
O6-MeG residues (18, 19), cisplatin G-G
intrastrand cross-links (18), adriamycin (20) and acetyl-aminofluorene
and aminofluorene (AAF/AF) adducts (21), and
S6-methylthioguanine/base mismatches (22). In
particular, recombination of nonreplicating UV-irradiated phage A recent study by Mu et al. (25) has provided direct
evidence for binding of human MSH2·MSH6 heterodimers (conveniently abbreviated hMutS We suggest that where error-prone DNA synthesis past UV photoproducts
produces photoproduct/base mismatches, mismatch repair directed against
error-rich nascent DNA by the usual strand-specificity signals might
remove most of these potential mutations, before fixation by nucleotide
excision repair or further semi-conservative replication. A logical
corollary is that mismatch repair should be highly specific for
photoproduct/base mismatches, because repeated initiation of repair at
matched photoproduct/base pairs might engender futile cycles that
wasted cellular energy and/or led to cell death (19, 27), and excision
gap-filling DNA synthesis past originally matched photoproducts might
introduce new mutations.
Here we have analyzed interactions of highly active purified hMutS We show here that hMutS Cell Extracts and Proteins--
HeLaS3 cells were either
purchased from the National Cell Culture Center (Minneapolis, MN) or
grown in Joklik modified medium (Life Technologies Inc.) supplemented
with 5% calf serum, at the Cell Culture Facility, Oregon State
University Environmental Health Science Center. Nuclear extracts were
prepared from HeLaS3 cells as described (26). In brief, proteins were
extracted with 0.15 M salt from nuclei released from the
cells broken by homogenization and then precipitated with ammonium
sulfate. hMutS Preparation of DNA 51-mer Top Strands Containing UV
Photoproducts--
All oligonucleotide sequences used here were
designed to be free from significant secondary structure, using CPrimer
program (free software from Regents of University of California). DNA 11-mers containing no photoproducts
(Table I, oligomers 1 and 4) or single
site-specific T<>T, T(6-4)T, or U<>U photoproducts (oligomers 2, 3, and 5) were prepared and purified by high pressure liquid
chromatography as described (28-30). The U<>U oligomers were then
5'-phosphorylated using phage-T4 polynucleotide kinase and unlabeled
ATP. (The T<>T and T(6-4)T oligomers were purified in phosphorylated
form.) Chromatographic analyses indicated that T<>T, T(6-4)T, and
U<>U oligomers were >99.5, >99, and >95% pure, respectively. The
"top right" flanking 20-mer 7 was purchased from Life Technologies,
Inc., phosphorylated, and high pressure liquid chromatography-purified.
The "top left" flanking 20-mer 6 and the bottom "scaffold"
41-mer 8 were synthesized at the Oregon State University Central
Services Facility, purified by electrophoresis in 20 or 10% denaturing
gels (polyacrylamide gels containing 7 M urea), and
recovered from gel by standard procedures (34). To prepare top strand
51-mers (oligomers 9-13), we used about 2.5 nmol each of oligomers
1-5, respectively. The latter were mixed with oligomers 6-8 (1.5
Single-strand 51-mers containing T<>T or U<>U CPDs were efficiently
cleaved by phage T4 endonuclease V, whereas T (6-4)T 51-mers were
completely resistant to endonuclease V but were digested by the 3'-5'
exonuclease activity of T4 DNA polymerase up to the photoproduct
positions, yielding 5'-end-labeled 26- and 25-mers.
Preparation of Duplex 51-mers--
Bottom strand 51-mers
(oligomers 14-18) were synthesized and purified as described above for
oligomer 8. These were mixed with various photoproduct-containing and
non-photoproduct top 51-mers in Annealing Buffer (10 mM
Tris-HCl, pH 8.0; 1 mM Na2EDTA; 100 mM NaCl), heated at 85 °C for 5 min, and slowly cooled
to room temperature. After addition of 0.2 volume of BND-cellulose (equivalent to 0.1 volume of settled resin), 5 M NaCl was
added to a final concentration of 1 M. Mixtures were
incubated 5 min and then layered onto Sephadex G-50 Nick Spin Columns
(Amersham Pharmacia Biotech, Uppsala, Sweden) that had previously been
equilibrated in DNA Buffer (10 mM Tris-HCl, pH 8.0; 1 mM Na2EDTA). Duplexes were recovered after
centrifugation, according to the instructions of the manufacturer. To
test for removal of single-stranded DNA, a small aliquot was treated
with polynucleotide kinase (which prefers 5'-single-stranded DNA ends
(35)) and [ Electrophoretic Mobility Shift Competition Assays--
Reaction
mixtures (25 µl) contained 1.32 nM
32P-5'-end-labeled TT/AG (oligomers 9/16) duplex 51-mers in
Binding Buffer (10 mM Hepes-KOH, pH 7.5; 110 mM
KCl; 1 mM dithiothreitol; 0.4% glycerol), plus particular
competing 51-mer duplexes at various concentrations, and hMutS Direct Binding Experiments--
Titrations with hMutS Binding Properties of hMutS Relative Affinities of hMutS
Since cytosines in cyclobutane-pyrimidine-dimers deaminate much more
rapidly than non-photoproduct cytosines (32), T<>U, U<>T, and
U<>U photoproducts might be expected to accumulate in UV-irradiated
cells. Where deamination occurred in the vicinity of appropriate
signals for initiation of excision, mismatch repair might thus actually
fix mutations. We measured the affinity of hMutS Effect of ATP on Binding of hMutS We have used equilibrium binding studies to address the hypothesis
that eukaryotic MSH2·MSH6 heterodimers, such as the hMutS Nevertheless, hMutS The apparent low affinity of hMutS To test the notion that hMutS The results presented here show that one prerequisite for efficient
correction by long-patch mismatch repair of mutations introduced in
human cells by error-prone translesion synthesis, namely specific
recognition of photoproduct/base mismatches by hMutS). Both (cyclobutane-dimer)/AG and
((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of
photoproduct/(A(T or C)) mismatches also showed higher hMutS
affinity than photoproduct/AA "matches"; the apparent affinity of
hMutS
for the ((6-4)photoproduct)/AA-"matched" substrate was
actually less than that for TT/AA homoduplexes. Surprisingly, although
hMutS
affinities for both non-photoproduct UU/GG double mismatches
and for (uracil-cyclobutane-dimer)/AG single mismatches were high,
affinity for the (uracil-cyclobutane-dimer)/GG mismatch was
quite low. Equilibrium binding of hMutS
to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced
similarly by ATP (in the absence of magnesium).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1000
fold, by recognizing and correcting mismatches that escape
proofreading by DNA polymerases (1). In Escherichia coli
(2), homodimers of MutS protein bind preferentially to mismatches; MutS
and MutL homodimers then activate MutH protein to nick specifically the
unmethylated DNA strand at the nearest adenine-hemimethylated d(GATC)
sequence, during the interval before adenines in newly replicated
d(GATC) sequences are methylated. This most likely involves a
translocation/search process that requires ATP hydrolysis (3). MutH
thus specifically directs incision and subsequent excision to the
nascent DNA strand, so replicative errors are always corrected rather
than fixed. After excision via exonucleolytic cleavage from the initial
incision past the mismatch, the replicative polymerase (here E. coli DNA polymerase III) fills the gap. The
MutH/d(GATC)-hemimethylation strand-specificity mechanism is not found
in eukaryotes or in some bacteria, and repair directionality remains
poorly understood in these organisms.
DNA
in (unirradiated) E. coli mutants deficient in excision
repair (UvrA
) was found to depend mostly on E. coli MutS, MutL, and MutH functions, on the presence of
unmethylated d(GATC) sequences in the phage DNA (16), and on helicase
and exonuclease functions previously implicated in mismatch repair
(17). Analysis of intracellular DNA revealed extensive
MutS-dependent breakdown of the nonreplicating UV-irradiated DNA (17). Studies of transcription-coupled nucleotide excision repair of cyclobutane-pyrimidine-dimers (CPDs) in E. coli and in human cells have, respectively, implicated MutS and MutL (23) and MSH2 and MLH1 (24) functions in these processes.
(26)) to DNA-containing UV photoproducts (or
cisplatin lesions). Binding to DNA-containing CPD/base mismatches (T<>T/AG) was roughly 1/3 as high as binding to non-photoproduct base/base mismatches (TT/AG). The marginal binding to matched CPDs
(T<>T/AA) and (6-4)photoproducts (T(6-4)T/AA) could not be clearly
distinguished from the nonspecific background. No data for (T(6-4)T/AG)
substrates were reported. In parallel experiments, T<>T/AG moieties
proved to be markedly better substrates for nucleotide excision repair
than T<>T/AA.
protein with both T<>T and T(6-4)T substrates. We tested all four
bases opposite the 3'-thymine in these photoproducts, the site of most
reported UV-induced mutations (Refs. 28-30 and reviewed in Ref. 31).
We also tested a series of U<>U substrates for binding by hMutS
,
because mismatch repair of Py<>U/PuG moieties arising by rapid
deamination of cytosines in CPDs (32) might actually fix mutations,
perhaps contributing to the surprising preponderance of C to T over T
to C transitions in UV-induced mutation spectra (31). Sensitivity was
high in these measurements, because of the absence of carrier DNA and
the low nonspecific background. To maximize resolution, we tested the
ability of the various photoproduct pairs to compete with a
radiolabeled T/G substrate, rather than directly measuring binding by
hMutS
.
binds with high specificity to both T<>T
and T(6-4)T photoproduct/base mismatches; binding to T(6-4)T/AA moieties actually appears less strong than to TT/AA homoduplexes. Although hMutS
recognizes all photoproduct/base mispairs, the highest affinity is for guanine opposite the 3'-thymine in either photoproduct. Affinity for single mismatches opposite U<>U
photoproducts is at least as high as for mismatched T<>T photoproducts.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein heterodimers were prepared essentially as
described (21, 26). Briefly, the 30-65% ammonium sulfate fraction
from HeLaS3 nuclear extracts was passed through a single-stranded
DNA-cellulose column, and the fraction subsequently eluted with 1 mM ATP, then chromatographed on a 1-ml Amersham Pharmacia
Biotech HR 5/5 Mono Q column. Purified hMutS
was supplemented with
1.0 mg/ml bovine serum albumin (BSA) and 10% (w/v) sucrose, frozen in
liquid nitrogen, and stored at
80 °C. SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining indicated that the purified
protein was near homogeneity (>98% pure). This procedure yielded
30-40 µg of protein from 10 to 15 liters of cells, as
determined by the Bradford assay using BSA standards
(33).
,
1.5
, and 2
fold excess, respectively) in DNA Ligase Buffer (50 mM Tris-HCl, pH 7.5; 10 mM MgCl2;
10 mM dithiothreitol; 1 mM ATP; 25 µg/ml BSA)
and heated at 85 °C for 5 min; after slow cooling to 16 °C,
mixtures were incubated overnight with 15 units of DNA ligase (New
England Biolabs, Beverly, MA). The product 51-mer top strands were
separated from shorter oligomers by electrophoresis in 10% denaturing
gels and recovered as described above.
Oligonucleotides used in this study (5'-3')
-32P]ATP (3000 Ci/mmol, NEN Life Science
Products) and electrophoresed in non-denaturing 10% polyacrylamide
gels. If necessary, the BND-cellulose and spin-column steps were
repeated. No single-stranded DNA contaminants were apparent in the
final autoradiographs. Conductivity of spin-column pass-through
solutions were found to be the same as for DNA buffer, i.e.
essentially all NaCl had been removed. Final DNA concentrations (typically 0.25-0.5 µM) were determined by the
Hoechst-33258 dye-binding fluorometric technique of Labarca and Paigen
(36), using a series of DNA standards and a TKO 100 Fluorometer (Hoefer
Scientific Instruments) according to the instructions of the
manufacturer. We prepared 32P-end-labeled non-photoproduct
T/G heteroduplexes similarly, except that the top 51-mer 9 was
32P-5'-end-labeled before mixing with bottom 51-mer 16.
protein at 3 nM. After incubation for 20 min on ice, mixtures were supplemented with 5 µl of 50% sucrose (w/v) in Binding Buffer, immediately loaded onto 6% polyacrylamide gels (36:1
acrylamide:bisacrylamide (w/w); 6.7 mM Tris acetate, pH
7.5; 1 mM Na2EDTA), and electrophoresed at 8 V/cm for 240 min, with recirculation of room temperature buffer. Gels
were dried onto Whatman 3MM paper and visualized by autoradiography at
room temperature, using Kodak Biomax x-ray film with intensifying
screens. To determine relative amounts of bound (mobility-shifted) and
free [32P]DNA, we measured band intensities using a
Molecular Dynamics PhosphorImager. We define fractional binding
FB(c) = (intensity of shifted
[32P]DNA band)/(total intensity of all
[32P]DNA bands), in the presence of competitor DNA at
concentration c, and we define c50 by
FB(c50)/FB(0) = 0.5. We determined c50 values for the various
competitors from straight lines fitted to
(FB(c))
1 versus
c plots; correlation factors for the fits were >0.97.
of
32P-labeled TT/AG 51-mer (prepared as described above), on
ice and at 37 °C, were performed as described under
"Electrophoretic Mobility Shift Competition Assay," except that
protein concentrations were varied from 0.2 to 5 nM and no additional competitor DNA was present. For testing effects of ATP on
direct equilibrium binding of hMutS
to TT/AG, T<>T/AG, and
T(6-4)T/AG 51-mers, respective unlabeled competitor duplexes were
end-labeled using overnight incubation with polynucleotide kinase plus
[
-32P]ATP, and higher ATP:DNA ratios than employed
with single-stranded DNA, then purified using Sephadex G-50 spin
columns. Mixtures containing 1.32 nM 51-mers, 3 nM hMutS
, and various concentrations of ATP were
incubated 20 min on ice, and then analyzed as described under
"Electrophoretic Mobility Shift Competition Assays," except that no
competitor DNA was present.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Protein--
Because we expected
that hMutS
might bind relatively weakly to some photoproduct base
pairs, we sought to maximize the sensitivity of binding assays. Use of
purified hMutS
of high specific activity minimized both spurious
bands due to binding by protein fragments and nonspecific binding by
partially inactive protein, making unnecessary the excess carrier DNA
employed in some previous studies. Free single-stranded DNA, for which
hMutS
shows significant
affinity,2 was completely
removed by BND-cellulose chromatography, as described under
"Experimental Procedures." These precautions made it possible to
accurately determine absolute concentrations of both bound and unbound
DNA. We initially tested the hMutS
preparation by using it to
titrate radiolabeled DNA 51-mers incorporating a T/G mismatch,
typically the base mispair recognized best by MutS-like proteins (26,
37, 38), employing electrophoretic mobility shift assays (Fig.
1A). Previous workers have
performed such assays at various temperatures. Here titrations at 4 and
37 °C yielded nearly identical binding curves (Fig. 1B).
Saturation was at 88 and 93% DNA binding, respectively, and apparent
half-saturation was at 1.3 nM hMutS
. Only negligible
levels of complexes moving faster than the major band were apparent
(Fig. 1A). To eliminate the possibility that the major band
corresponded to highly cooperative binding of two hMutS
heterodimers, which would result in a sigmoidal binding curve, we
measured binding at numerous low protein concentrations. The data (Fig.
1B) correspond to a strictly hyperbolic (non-cooperative) curve. Calculation of dissociation constants (Kd
values) from concentrations of bound and unbound DNA at hMutS
concentrations below 2 nM yielded 1.2 nM. A
similar titration experiment (data not shown) yielded a half-saturation
concentration of 1.3 nM. The apparent Kd
of 1.2 nM for this hMutS
preparation is at the extreme
lower range of values reported for previous binding studies. Previous
studies showed apparent saturation at various hMutS
:DNA ratios as
follows: 2-4 for hMutS
purified from HeLa cells in the presence
(26) or absence (21) of (20-fold) competitor, and 40-50 (18, 25), or
more than 600 (15) for recombinant proteins. The high concentrations of
nonspecific carrier DNA employed in some of these cases might have
affected apparent hMutS
binding properties. At hMutS
concentrations above 2 nM, increasing levels of a slower
moving complex appeared, corresponding to about 8% of bound DNA at the
highest protein concentrations. This might reflect nonspecific binding
of a second hMutS
molecule to regions of the 51-bp substrate outside
of the 25-35-bp region expected to be covered by a single heterodimer
bound to the central mismatch (15). The titration curve does not extend
far enough to determine unequivocally whether binding of this
additional protein is cooperative, i.e. facilitated by the
presence of the mismatch-bound hMutS
, or essentially independent.
The apparent absence of such multi-protein complexes in previous
studies may have been due to the smaller size of the heteroduplex
substrates employed and/or the presence of excess carrier DNA.
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Fig. 1.
Titration of (G/T) DNA with
hMutS protein. Mixtures of 1.32 nM [32P]5'-end-labeled (T/G) 51-mer and
indicated (nM) concentrations of purified hMutS
protein,
both prepared as described under "Experimental Procedures," were
incubated at 4 or 37 °C, and binding was assayed as described under
"Direct Binding Experiments." A, autoradiograph for
4 °C experiment. B, plot of fraction DNA bound (migrating
with reduced electrophoretic mobility) as determined by PhosphorImager
analyses. 4 °C (
) and 37 °C (
).
for Base-Base and Photoproduct-Base
Mismatches--
To maximize sensitivity to small differences in
hMutS
affinity among different substrates, we compared the abilities
of various unlabeled DNA 51-mers to compete with 1.32 nM
radiolabeled (T/G)-51-mer in mobility shift assays (in the absence of
other nonspecific carrier DNA), rather than directly measuring binding
affinities. Fig. 2A shows
typical competition analyses, for unlabeled 51-mers containing a TT/AG
base/base mispair (identical to the radiolabeled probe), a T<>T/AG
(cyclobutane-pyrimidine-dimer)/base mispair, and a T(6-4)T/AG (T(6-4)T)
(6-4)photoproduct)/base mispair. Binding to radiolabeled T/G 51-mer was
reduced 50% by an equal concentration of unlabeled T/G 51-mer, as
expected. Fig. 2B shows a typical plot of the reciprocal of
the fractional binding of radiolabeled (T/G)-51-mer by hMutS
at
various competitor concentrations, relative to binding in the absence
of competitor. The concentrations corresponding to 50% reduction in
binding (ordinate value of 2.0 in Fig. 2B), for a variety of
substrates with TT, T<>T, or T(6-4)T upper strands, are summarized in
Fig. 3. Affinities relative to TT/AA
homoduplexes appear in parentheses. Several results are of particular
interest. First, all base-base mismatches analyzed here were
specifically bound by hMutS
, consistent with previous binding
studies (26, 37, 38) and with observations that all eight base-base
mismatches are efficiently repaired in cell-free extracts (39) (and
apparently in vivo (40)). The trend in affinity order was
TT/AG > TT/AC > TT/AT > TT/AA. Second, all mismatched
substrates involving cyclobutane-pyrimidine-dimers showed greater
affinity for hMutS
than the "matched" T<>T/AA substrate, for
which significant Watson-Crick character is expected (41, 42). The
trend in affinity was similar to that for base-base mismatches.
Although the apparent T<>T/AG and T<>T/AC affinities were only
about 1/8 the affinity of the TT/AG base/base mismatch, both
affinities are as high as that of the TT/AT base/base mismatch, which is repaired efficiently in vitro (39) and in
vivo (40). However, relative affinities and repair
efficiencies may be affected by sequence context. Third, mismatched
(6-4)photoproduct substrates showed considerably more affinity than the
matched T(6-4)T/AA substrate, although both photoproduct base
combinations are expected to be highly distorted and devoid of hydrogen
bonding at the 3'-photoproduct base (43). Again, T(6-4)T/AG showed the
highest affinity. The apparent affinity of a 51-mer containing a single
T(6-4)T/AA moiety was actually significantly less than that of the
TT/AA homoduplex (p = 0.0056, based on analysis of
variance and pairwise t tests of data for the three matched
substrates). The mismatch affinity ratios, i.e. ratios
of affinities for TT/AG, T<>T/AG, and T(6-4)T/AG mismatches
relative to affinities for the corresponding matched substrates, were, respectively, 20, 3.6, and 4.3.
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Fig. 2.
Competition analysis of
hMutS binding to mismatched DNA
substrates. Mixtures of 1.32 nM
32P-5'-end-labeled (T/G) 51-mer, a particular concentration
of a particular photoproduct/base or base/base mismatched 51-mer, plus
3 nM hMutS
, purified as described under "Experimental
Procedures," were analyzed as described under "Electrophoretic
Mobility Shift Competition Assays." A, representative
autoradiograph for binding of radiolabeled TT/AG 51-mer in the presence
of indicated concentrations of unlabeled base/base and
photoproduct/base 51-mers. B, plots (data from A)
of the fraction of DNA bound (amount of [32P]DNA showing
altered mobility divided by total [32P]DNA) in the
absence of unlabeled competitor, divided by the fraction bound at
indicated concentrations of unlabeled TT/AG 51-mer (
), T<>T/AG
51-mer (
), or T(6-4)T/AG 51-mer (
). Indicated straight lines were
fitted to the respective data sets.
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Fig. 3.
Competition for hMutS
binding by DNA containing thymine/base or
(photoproduct-thymine)/base mismatches. Binding/competition
experiments, similar to those shown and analyzed in Fig. 2, were
performed using 1.32 nM 32P-5'-end-labeled T/G
51-mers, 3 nM hMutS
, and 0.33 to 84 nM
concentrations of indicated unlabeled competitor 51-mers. Competitor
concentrations corresponding to 50% reduction in binding (ordinate
value of 2.0 in fitted plots similar to those in Fig. 2B)
are shown. Mismatch affinity factors (reciprocals of ratios of
concentrations showing 50% reduction relative to concentration for
TT/AG (27.8 nM)) are indicated in parentheses.
Individual symbols (
,
, and
) correspond to independently
determined competition curves.
for
uracil-containing base/base and photoproduct/base single and double
mismatches (Fig. 4). Among base/base
mismatches, UU/AG and UU/GG showed, respectively, somewhat less and
somewhat more affinity than TT/AG mismatches, relative to the
corresponding homoduplexes; the trend UU/AG > UU/AC > UU/AT
paralleled that for the TT series. Affinities of hMutS
for all three
single U<>U/base mismatches appeared higher than the affinity for
U<>U/AA; the mismatch affinity ratio for U<>U/AG was 6.7, considerably higher than the (T<>T/AG):(T<>T/AA) ratio of 3.6. Surprisingly, even though the UU/GG and U<>U/AG affinity ratios were,
respectively, 24 and 6.7, the ratio for the double (U<>U) base
mismatch was only 1.4.
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Fig. 4.
Competition for hMutS
binding by DNA containing uracil/base or
(photoproduct-uracil)/base mismatches. Binding/competition
experiments, similar to those shown and analyzed in Fig. 2, were
performed using 1.32 nM 32P-5'-end-labeled T/G
51-mers, 3 nM hMutS
, and 0.33 to 84 nM
concentrations of indicated unlabeled competitor 51-mers. Competitor
concentrations corresponding to 50% reduction in binding (ordinate
value of 2.0 in fitted plots similar to those in Fig. 2B)
are shown. Mismatch affinity factors relative to UU/AA (see legend to
Fig. 3) are indicated in parentheses. Individual symbols
(
and
) correspond to independently determined competition
curves.
to Base/Base and
Photoproduct/Base Mismatches--
An early step in mismatch-repair
pathways is thought to be ATP-triggered translocation/dissociation of
MutS-like proteins away from the mismatches to which they had been
bound (3, 14, 15). Consequently, the apparent equilibrium binding of
these proteins to relatively short linear mismatched oligomers is
highly sensitive to ATP (14, 15). If binding of hMutS
to
photoproduct/base mismatches is analogous to its binding to base/base
mismatches, ATP sensitivities should be similar. We tested the effect
of different ATP concentrations on direct binding of hMutS
to
radiolabeled TT/AG, T<>T/AG, and T(6-4)T/AG substrates (Fig.
5). The binding of hMutS
to the
photoproduct/base mismatches seen here in the absence of ATP confirms
our assumption that the reduction of hMutS
binding to T/G in the
presence of these photoproduct/base mismatches (Fig. 3) reflects the
direct binding affinities of the latter. The amount of T<>T/AG DNA
bound at this concentration of hMutS
(3 nM) is about 1/2
the amount of TT/AG 51-mer bound, in good agreement with the
measurements of Mu et al. (25). The ATP sensitivities for
these substrates were quite similar; estimated ATP concentrations at
half-maximal binding were, respectively, 58, 71, and 79 µM (Fig. 5). Since these experiments were performed in
the absence of magnesium, the results are consistent with previous
reports that ATP binding but not ATP hydrolysis is required for the
dissociation of hMutS
from mismatched duplexes (14, 15).
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Fig. 5.
Effect of ATP on binding of
hMutS to base/base and photoproduct/base DNA
mismatches. Binding of 1.32 nM
32P-5'-end-labeled TT/AG, T<>T/AG, and T(6-4)T/AG 51-mer
by 3 nM hMutS
at 4 °C, in the presence of indicated
concentrations of Na-ATP (in the absence of Mg2+) were
performed as described under "Direct Binding Experiments." The
fraction of DNA bound corresponds to the amount of
[32P]DNA showing reduced mobility divided by total
[32P]DNA in each lane. Means of three independent
experiments for TT/AG (
), T<>T/AG (×), and T(6-4)T/AG (
) and
standard deviations are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein
studied here, bind more strongly to photoproduct/base mismatches, such
as T<>T/AG, (6-4)T/AG, and U<>U/AG, than to the respective matched
photoproducts, T<>T/AA, T(6-4)T/AA, and U<>U/AA. Although our
studies have sensitively delineated relative affinities among an
extensive set of substrates, equilibrium binding cannot measure the
full quantitative range of biological specificity. The T/G 51-mer is
only 20 times as effective as a T/A substrate in competing for
hMutS
. T/C and T/T mismatches, known to be corrected efficiently
in vitro and in vivo, compete less well. Even if
there are occasional altered guanine bases in putative homoduplexes, due to chemical damage during oligonucleotide
synthesis,3 these should be
present at the same frequency in the T/G substrate and the
photoproduct-containing substrates, so the effects of any altered bases
should to a first approximation cancel out in the competition
experiments. The single mismatches in these 51-mer substrates are
diluted by 50 matched base pairs, but during replication of the human
genome, hMutS
(or hMutS
) protein must initiate correction of
perhaps one or two thousand mismatches, scattered among billions of
homoduplex pairs. Any significant degree of mismatch-repair
incision/excision directed against homoduplex "false positives"
would clearly be highly deleterious to the cell. Postulated
post-binding steps in the pathway, such as repeated cycles of ATP
binding and hydrolysis coupled to hMutS
translocation (3, 14, 15),
or ATP-dependent protein recruitment and dissociation from
the mismatch (15), or directed helicase/exonuclease action (39, 44),
might be more specific, or the initial hMutS
search/recognition process in vivo might be more complex (and specific) than
simple equilibrium binding.
shows a clear preference for photoproduct/base
mismatches. The mismatch affinity factors of 3.6 and 4.3 for T<>T/AG
and T(6-4)T/AG (relative to the respective photoproduct/base matches)
are less than the factor of 20 for TT/AG (relative to TT/AA) but
greater than the factor (2.4) for a non-photoproduct TT/AT mismatch.
Since significant hydrogen bonding and Watson-Crick character are
retained at the 3' (and 5') position(s) in CPDs (41, 42), it
is perhaps not surprising that preferences for recognition of various
CPD/base mismatches by hMutS
here roughly parallel the affinity
order for base/base mismatches, which is in fact the same order as seen
for repair of all eight base/base mismatches in vivo (40).
However recognition of (6-4)photoproduct mismatches was unexpected.
These are much more distorted, especially at the 3' position, where
there appears to be no hydrogen bonding (43), and an extracyclic oxygen
is actually shifted from the 3'- to the 5'-thymine. It is far from
clear how a protein binds T(6-4)T/AG well but T(6-4)T/AA weakly, when
the 3'-photoproduct is so poorly instructive. It may be significant
that the affinity trend for intermediate substrates, T(6-4)T/AT > T(6-4)T/AC, does not parallel the base/base and CPD/base trends.
for T(6-4)T/AA, even lower than
for TT/AA homoduplexes, hints at complexity in the recognition process.
If hMutS
repeatedly searches for mismatches by transiently binding
one or a few base pairs in a recognition site, then dissociating when
no mismatch is found, a single T(6-4)T/AA moiety among 50 Watson-Crick
pairs would seem unlikely to increase appreciably the average rate of
dissociation. However, recognition might involve unidirectional or
bi-directional one-dimensional scanning of DNA by hMutS
until a
mismatch is recognized and bound. Even in the absence of a mismatch,
the loose DNA-protein affinity during the scanning process might cause
significant reduction in DNA electrophoretic mobility. However, if
hMutS
were to dissociate completely when it encountered a T(6-4)T/AA
moiety, scanning would be disrupted, and there would be no mobility
reduction until hMutS
reinitiated the process.
recognizes mismatched bases opposite
deaminated 3'-cytosines in Py<>C CPDs, leading to fixation rather
than correction of these potential mutations, we employed a series of
U<>U/AN substrates. (It seems likely that T<>U/AN moieties would
show similar results.) Specific recognition of the series of
3'-mismatched U<>U CPDs by hMutS
paralleled recognition of the
T<>T series; U<>U/AG, a model for mismatch created by deamination, was the best substrate. We also used U<>U substrates to model the
product of double deamination, predicted to be significant in
vivo (32), which might contribute to the high frequency of CC to
TT transitions found in mutation spectra (31). Surprisingly, although
non-photoproduct UU/GG double mispairs were bound twice as well as
UU/AG single mispairs, no specific affinity for the U<>U/GG double
photoproduct/base mismatches was observed. Perhaps the extent of helix
distortion/opening here exceeds the hMutS
recognition capability, as
appears to be the case for large insertion/deletion loop-outs (6).
protein, is
met. It remains to be determined whether the repair synthesis
associated with mismatch repair bypasses the same lesions with
reasonable efficiency and fidelity. If so, there would be at least
three outcomes after misinsertion opposite photoproducts during
semi-conservative replication, the first two resulting in mutation
fixation: (i) T<>T/AG to T<>T/AA + TC/AG by replication; (ii) T<>T/AG
to TC/AG by excision repair; (iii)
T<>T/AG to T<>T/AA by mismatch repair, thus preventing
mutation. In case iii, mutation avoidance might therefore proceed by
Steps 1-3 as follows.
(Step 1)
(Step 2)
Alternatively, blocked repair synthesis might lead to cell death,
apparently an important form of mutation avoidance in mammalian cells,
and/or recombinational bypass using presumably error-free sister-chromatid DNA. It is now clearly of high interest to determine whether MutS-like proteins in other organisms show the same specificity and whether UV mutagenesis is elevated in prokaryotic and/or eukaryotic mismatch-repair-deficient mutants.
(Step 3)
Recent work in our laboratory demonstrates that UV-induced
CCC to TCC and TTC to TCC
revertant frequencies at lacZ codon 461 are elevated in
E. coli mutS, mutL, and mutH mutants,
significantly above the higher spontaneous frequencies in these
strains.4 This result is
consistent with the hMutS specificity documented here, but the
previously described MutHLSdependent recombination of
nonreplicating UV-irradiated d(GATC)-undermethylated phage
DNA (16)
now appears paradoxical. Newly irradiated
virions would be expected
to contain far less than one base/base mismatch per (50 kilobase pairs)
genome and essentially zero photoproduct/base mismatches. If E. coli MutS, unlike hMutS
, were to initiate mismatch repair in
response to matched as well as mismatched photoproducts, then the
apparent antagonism of E. coli UV mutagenesis by mismatch repair cited would imply virtually error-free filling of excision gaps,
perhaps by efficient sister-chromatid exchange. Alternatively, deamination of cytosine-containing photoproducts in the nonreplicating UV-irradiated phage DNA might be accelerated in vivo, by an
unknown mechanism, such that recombinagenic Py<>U/PuG or
U<><Py/GPu mismatches appeared rapidly enough to stimulate
recombination as early as 60 min after infection (16).
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ACKNOWLEDGEMENT |
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We thank Dr. Clifford Pereira, Environmental Health Sciences Center and Department of Statistics, Oregon State University, for advice on graphical presentations and statistical analysis.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant RPG-96-074-03-CNE (to J. B. H.), and some initial studies were supported by National Institutes of Health Grant CA 72956 (to G.-M. L.). This is Technical Paper 11490 from the Oregon Agricultural Experimental Station.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.
To whom correspondence should be addressed. Tel.:
541-737-1777; Fax: 541-737-0497; E-mail: haysj{at}bcc.orst.edu.
2 H. Wang and J. Hays, unpublished observations.
3 P. Modrich, personal communication.
4 H. Liu, S. Hewitt, and J. Hays, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
MSH, MutS homologs;
bp, base pairs;
BSA, bovine serum albumin;
CPD, cis-syn-cyclobutane-pyrimidine-dimers;
(6-4)photoproduct, pyrimidine-(6-4')pyrimidinone photoproduct;
hMutS, human MSH2·MSH6
protein heterodimer;
T<>T/AA, T<>/AG, T<>/AC, T<>T/AT,
C<>C/GG, U<>U/AA, U<>U/GG, U<>U/AG, U<>U/AC, U<>U/AT,
thymine-, cytosine-, or uracil-containing cyclobutane-dimers (5'
3' as
shown), appearing in DNA opposite indicated (3'-5') nucleotides;
T(6-4)T/AA, T(6-4)T/AG, T(6-4)T/AC, T(6-4)T/AT, (thymine-containing)
(6-4)photoproducts (shown 5'
3') appearing in DNA opposite indicated (3'
5') dinucleotides;
Pu, purine nucleotide;
Py, pyrimidine
nucleotide;
BND-cellulose, benzoylated naphthoylated
DEAE-cellulose.
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