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INTRODUCTION |
The short-patch MutY repair pathway of Escherichia coli
specifically repairs A/GO and A/G to C/GO and C:G, respectively, and corrects A/C to G:C at a much lower rate (1-7). The MutY protein along
with MutM and MutT is involved in defending against the mutagenic
effects of 7,8-dihydro-8-oxo-guanine (8-oxoG or
GO)1 lesions, the most stable
products formed due to oxidative damage to DNA (8, 9). The MutT protein
eliminates 8-oxo-dGTP from the nucleotide pool with its nucleoside
triphosphatase activity (10-12). The MutM glycosylase (Fpg protein)
provides a second level of defense by removing both ring-opened purine
lesions and mutagenic GO adducts (13, 14). When C/GO is not repaired by
MutM, adenines are frequently incorporated opposite GO bases during DNA
replication (15, 16). A second round of replication through this
mismatch subsequently leads to a G:C to T:A transversion (16-19). MutY
provides a third level of defense by removing the adenines
misincorporated opposite GO or G following DNA replication (3, 8,
20).
The MutY protein is a 39-kDa iron-sulfur protein (7, 21, 22) with its
N-terminal domain sharing structural similarity with endonuclease III
(endo III) and AlkA (7, 21-24). This includes the helix-hairpin-helix
and Gly/Pro ... Asp loop motifs. Endonuclease III repairs thymine
glycol and oxidized pyrimidines in DNA (25, 26) and AlkA repairs
methylated purines (27, 28). The conserved Asp acts as a general base
to activate a nucleophile such as Lys or water. DNA glycosylases in the
endo III superfamily can be divided into two groups (29, 30).
Bifunctional DNA glycosylases, including endo III and OGG1, use the
conserved lysine (Lys120 in endo III and Lys249
in hOGG1) to form a Schiff base intermediate and also possess strong AP
lyase activity (23, 31-33). Monofunctional glycosylases such as AlkA
lack both the conserved lysine and AP lyase activity (27, 28, 34).
Because MutY does not have the conserved lysine downstream of the
helix-hairpin-helix motif (Lys120 in endo III and
Lys249 in hOGG1), it was originally grouped as a
monofunctional glycosylase (34, 35) in the endo III superfamily. The
possession of apurinic/apyrimidinic (AP) lyase activity by MutY is
controversial. The MutY AP lyase activity was detected by some groups
(7, 20, 36-40) but not by other groups (3, 4, 34, 35, 41). However,
there is supporting evidence that MutY may be a bifunctional
glycosylase. MutY can form a covalent complex with its DNA substrates
(37-39, 42, 43), a diagnostic tool for bifunctional glycosylase/AP lyases (29, 33, 34). Several reports have shown that the Schiff base
intermediate formation involves Lys142 (43-45), however,
the K142A mutant MutY has glycosylase activity (44, 45) and can promote
a 
/
-elimination on AP-containing DNA (45). MutY can also cleave
DNA containing an unmodified AP site (37, 45). Thus, MutY represents a
unique group of glycosylases.
The structure and function of MutY have revealed several interesting
aspects. Ethylation interference studies showed that MutY interacts
with at least five phosphate groups and covers about 12 base pairs
around the A/G mismatch (20). Methylation interference experiments have
demonstrated that MutY specifically interacts with both mispaired A and
G as well as the two bases flanking the A/G mismatch (20). This may
explain the neighboring sequence effect on the repair efficiency of
MutY. The MutY domain structure has been studied through proteolysis
(37, 39, 40) and site-directed mutagenesis (24, 38, 46). The N-terminal domains of MutY, residues 1-226 (M25) (39, 47), and residues 1-225
(cdMutY or p26) (37, 40) have been shown to retain catalytic activity.
Recently, the x-ray crystal structure of the catalytic domain of
MutY(D138N) (cdMutY) with bound adenine shows that the adenine is
buried in the active site of the catalytic domain and suggests that the
mismatched adenine must flip out of the DNA helix for the glycosylase
action (24). In the active site pocket, several amino acids
(Gln182, Glu37, and Asp186) are
involved in adenine binding.
The crystal structure of the N-terminal domains of MutY also suggests
some candidate residues for the recognition of the base opposite
adenine. We and Clarke's group (39, 47) have shown that the C-terminal
domain of MutY plays an important role in the recognition of GO lesions
while Manuel and Lloyd (37) have not drawn the same conclusion. In this
paper, we further investigated the role of the C-terminal domain of
MutY. The truncated MutY construct, M25, has more than 18-fold lower
affinities for binding 8-oxoG-containing mismatches tested, as compared
with intact MutY. Deletion of the C-terminal domain reduces its
catalytic preference for A/GO-DNA over A/G-DNA. MutY also exerted more
inhibition on the catalytic activity of MutM (Fpg) protein than M25.
Moreover, an E. coli mutY strain that produces an N-terminal
249-residue truncated MutY is a mutator. These findings strongly
support the notion that repair of 8-oxoG is the major function of MutY
and its 8-oxoG specificity is located mainly in the C-terminal domain.
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EXPERIMENTAL PROCEDURES |
E. coli Strains--
E. coli PR8
(Su
lacZ X74 galU galK Smr), PR70
(like PR8 but micA68::Tn10Kan), and
PR68 (like PR8 but micA68::mini Tn10Kan
mutL218::Tn10) were obtained from
M. S. Fox.
Cloning of the N-terminal Domain of mutY Gene--
The
N-terminal domain of the mutY gene corresponding to
Met1 to Gln226 (M25) was polymerase chain
reaction amplified from the pJTW10-12 plasmid (7) using primers
Chang 222 (5'-GCGACGCATATGCAAGCGTCGCAATTTTC-3') and Chang 223 (5'-GCCGGAGGATCCCTACTGTTTCGGTTTTTTGCCCG-3'). Primer Chang 222 contains an NdeI site at the 5' end of the coding sequence and primer Chang 223 contains a BamHI site after the stop
codon. The polymerase chain reaction product was purified from agarose gels, cut with NdeI and BamHI, and cloned into an
NdeI/BamHI-digested pET11a expression vector. The
inserted MutY sequence in the resulting clone pJ16-146-13
was confirmed by DNA sequencing. The expression of M25 was under the
control of the T7 promoter. The expression host of the mutY
mutants, PR70 harboring the 
DE3 lysogen, was constructed according
to the procedures described by Invitrogen.
Expression and Purification of M25 and MutY--
E.
coli strain PR70/DE3 harboring the expression plasmid containing
M25 was grown in LB broth containing 50 µg/ml ampicillin at 37 °C.
The expression of the protein was induced at an
A590 of 0.6 by the addition of
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 0.4 mM to the culture at 20 °C. The cells were harvested 16 h later.
The M25 domain was purified from 40 g of E. coli
PR70/DE3 cells harboring the overproduction plasmid, similar to the
method used with wild-type enzyme (7). Nicking of A/G-containing 20-mer DNA was monitored during the M25 protein purification. As judged on a
12% SDS-polyacrylamide gel, M25 was purified to >99% homogeneity (data not shown). The purification of homogeneous MutY protein from an
overproducing E. coli JM109 strain, harboring pJTW10-12, has been described previously (7).
Expression and Purification of MutM--
The E. coli
mutM gene was polymerase chain reaction amplified from the
pFPG60 plasmid (7, 48) using primers Chang 180 (5'-GCCGGAATTCGGAGATGCTATGCCTGAATT-3') and Chang 181 (5'-GCCGGAATTCTTACTTCTGGCACTGCCGAC-3'). Both primers contain an
EcoRI site at the 5' end of the coding sequence or after the
stop codon. The polymerase chain reaction product was purified from
agarose gels, cut with EcoRI, and cloned into an
EcoRI digested pKK223-3 expression vector. Expression of
MutM protein was similar to that of M25 as mentioned above. MutM
protein was purified to more than 95% homogeneity from E. coli JM109 cells harboring the overproduction plasmid by ammonium sulfate precipitation, phosphocellulose, hydroxylapatite, and heparin
chromatographies similar to the method used with MutY enzyme (7).
Oligonucleotide Substrates and Enzymes--
The
nucleotide sequences of oligonucleotide pairs used in this study
were as follows: 19-mer 5'-CCGAGGAATTXGCCTTCTG-3' and 3'-GCTCCTTAAYCGGAAGACG-5' and 40-mer
5'-AATTGGGCTCCTCGAGGAATTXGCCTTCTGCAGGCATGCC-3'and 3'-CCCGAGGAGCTCCTTAAYCGGAAGACGTCCGTACGGGGCC-5' (X = A, C, G, T, I, or U; Y = C, G, or GO). Oligonucleotides of
19-mer and 40-mer containing base mismatches were labeled at the 3' or
5' ends as described by Lu et al. (20). After filling-in the
sticky ends with the Klenow fragment of DNA polymerase I, the 19-mer
and 40-mer were converted into 20-mer and 44-mer, respectively. DNA
substrates containing U/G or U/GO (300 fmol) were fully converted to
AP/G or AP/GO by treating with 1.5 units of E. coli uracil
DNA glycosylase (UDG, Life Technologies, Inc.) at 37 °C for 1 h
in MutY buffer (20 mM Tris-HCl, pH 7.6, 80 mM
NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 2.9% glycerol).
MutY Binding, Trapping, and Cleavage Assays--
The MutY
activity assays with labeled oligonucleotide substrates were performed
as described by Lu et al. (38) with some modifications. MutY
enzyme was diluted with diluent (20 mM potassium phosphate,
pH 7.4, 50 mM KCl, 1.5 mM dithiothreitol, 0.1 mM EDTA, 200 µg/ml bovine serum albumin, and 50%
glycerol) before use. The MutY binding reaction mixture contains 20 mM Tris-HCl, pH 7.6, 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 2.9% glycerol, 20 ng of poly(dI-dC), and 1.8 fmol of labeled DNA in a total volume of
10 µl. After incubation at 37 °C for 30 min, the mixtures were supplemented with 1.5 µl of 50% glycerol and analyzed on 8%
polyacrylamide gels in 50 mM Tris borate, pH 8.3, and 1 mM EDTA. To determine the Kd values,
nine different MutY enzyme concentrations were used to bind DNA
substrates and experiments were repeated at least three times. Bands
corresponding to enzyme-bound and free DNA were quantified from
PhosphorImager images and Kd values were obtained
from analyses by a computer-fitted curve generated by the Enzfitter
program (49).
Covalent complexes of M25 and MutY with 19-mer DNA substrates were
formed in a reaction containing 20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 1 mM EDTA, 2.9% glycerol,
and 0.1 M NaBH4. A NaBH4 (1 M) stock solution was freshly prepared and added
immediately after the enzyme was added. After incubation at 37 °C
for 30 min, 5× dye buffer (0.5 M sucrose, 15% SDS, 312.5 mM Tris-HCl, pH 6.9, 10 mM EDTA, 5%
-mercaptoethanol, and 0.0025% bromphenol blue) was added to the
samples, which were heated at 90 °C for 2 min and separated on a
12% polyacrylamide gel in the presence of SDS according to Laemmli
(50).
The glycosylase assays were carried out similarly to the trapping assay
except no NaBH4 was added. After incubation at 37 °C for
various times, reaction mixtures were lyophilized, resuspended in 3 µl of formamide dye (90% formamide, 10 mM EDTA, 0.1%
xylene cyanol, and 0.1% bromphenol blue), heated at 90 °C for 2 min, and loaded onto 14% 7 M urea sequencing gels. For
time course studies, after enzyme reaction at different time points,
samples were immediately frozen at 
70 °C, followed by heating at
90 °C for 30 min with 1 M piperidine, and then treated
as the glycosylase reaction.
MutM Cleavage of T/GO-, C/GO-, G/GO, and AP/GO-containing
Substrates--
The reaction mixture contained 1.8 fmol of duplex
20-mer DNA substrates and 0.09 nM MutM for C/GO, T/GO, and
G/GO mismatches and 0.36 nM MutM for AP/GO mismatch,
respectively, in the MutY buffer. The reaction was preincubated for 2 min at room temperature with increasing amounts of MutY, M25, or
diluent. MutM was then added, and the reaction was further incubated at
37 °C for 28 min. Samples after reactions were lyophilized,
resuspended in 3 µl of formamide dye, heated at 90 °C for 2 min,
and loaded onto 14% 7 M urea sequencing gels.
Western Blot Analysis--
Proteins were resolved on a SDS-12%
polyacrylamide gel and transferred to a nitrocellulose membrane (51).
The membrane was subjected to the Enhanced Chemiluminescence analysis
system from Amersham Pharmacia Biotech according to the manufacture's
protocol. The anti-MutY antibodies were affinity purified by reaction
with membrane-bound MutY protein (52).
Measurement of Mutation Frequency--
Independent overnight
cultures (0.1 ml) of each strain were plated onto LB agar containing
0.1 mg/ml rifampicin. The cell titer of each culture was determined by
plating a 106 dilution onto LB agar. For each
measurement, four independent cultures were plated and the experiments
were repeated three times. The ratio of RifR cells to total
cells was the mutation frequency.
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RESULTS |
G/GO Is a Substrate for MutY--
We have shown that MutY has much
tighter binding, but weaker glycosylase activity to A/GO- than
A/G-containing DNA at an enzyme/DNA molar ratio of 40 at 37 °C for
30 min in a steady-state kinetic study (38). To further delineate the
reactivity of MutY on different mismatches containing GO, we
constructed A/GO, C/GO, G/GO, and T/GO duplex DNA substrates. As shown
in Table I, the apparent dissociation
constants (Kd) of MutY with A/GO, G/GO, and T/GO are
comparable. MutY binding to C/GO-containing DNA was 170-fold weaker
than that with A/GO but only 2-fold weaker than that with
A/G-containing DNA.
The combined MutY DNA glycosylase/AP lyase activities can be assayed by
monitoring the nicking products in a denatured sequencing gel. As shown
in Fig. 1A, MutY adenine
glycosylase removed adenines from A/G and A/GO mismatches (Fig.
1A, lanes 2 and 4). DNA substrates containing
T/GO and C/GO were not cleaved by MutY (Fig. 1A, lanes 8 and
10). Surprisingly, the duplex containing G/GO was cleaved about 5% of the extent of DNA containing an A/GO mismatch (Fig. 1A, lane 6). A Schiff base intermediate between MutY and DNA
can be trapped in a stable enzyme-DNA covalent complex in the presence of sodium borohydride. At an enzyme:DNA molar ratio of 40, MutY could
trap A/G and A/GO mismatches efficiently (Fig. 1B, lanes 2 and 4) but not T/GO and C/GO (Fig. 1B, lanes 8 and 10). MutY could also trap the G/GO-containing DNA (Fig.
1B, lane 6) although its efficiency is much weaker (less
than 2%) than A/G- and A/GO-DNA (Fig. 1B, lane 6). Thus,
MutY is both an adenine and guanine DNA glycosylase.
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Fig. 1.
MutY glycosylase and trapping activities with
DNA substrates. 1.8 fmol of 3'-end labeled A/G (lanes 1 and 2), A/GO (lanes 3 and 4), G/GO
(lanes 5 and 6), T/GO (lanes 7 and
8), and C/GO (lanes 9 and 10)
containing 20-mer oligonucleotides were incubated for 30 min at
37 °C with 72 fmol of MutY (even lanes). Odd
lanes are DNA alone. A, glycosylase
activity. Glycosylase coupled with -elimination reaction
leads to strand cleavage. The products, after reaction were lyophilized
(without piperidine treatment), resuspended in formamide dye, heated at
90 °C for 2 min, and analyzed on a 14% denaturing sequencing gel.
Arrows indicate the positions of intact oligonucleotide
(I) and the nicked product (N). B,
trapping activity. Trapping reactions were performed in the
presence of 0.1 M NaBH4 and the products were
analyzed on an 8% SDS-polyacrylamide gel. The film was overexposed to
show the weak complex of MutY with G/GO mismatch. Arrows
indicate the positions of free and enzyme-bound
DNA substrates.
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DNA Mismatch Binding of M25 and MutY--
Previous results have
shown that the N-terminal domain (M25, Met1 to
Gln226), obtained from proteolysis of MutY or expressed as
a recombinant protein, retains the catalytic activity (39, 47). The
tight binding of A/GO mismatch by MutY is lost in M25, suggesting that the C-terminal region of MutY, a region that has no counterpart in
E. coli endo III, plays an important role in the recognition of A/GO mismatched DNA. To investigate the function of the C-terminal domain of MutY, the same truncated MutY fragment was overproduced and
purified to near homogeneity and its activity was compared with the
intact protein. Gel shifting DNA binding experiments demonstrated that
recombinant M25 had Kd values of 6.8, 34, and 4.3 nM with A/G-, A/C, and A/GO-containing 20-mer DNA substrates, respectively (Table I, first three rows). The results show
that M25 and MutY have similar binding affinities to A/G and A/C
mismatches. However, M25 had a 67-fold weaker binding affinity to
A/GO-containing DNA than did intact MutY. These results with expressed
M25 are consistent with the previous findings of Gogos et
al. (39) and Noll et al. (47).
The truncated and intact MutY also had different binding affinities
with other mismatches containing GO. As shown in Table I, intact MutY
had more than 18-fold higher binding affinities with A/GO, I/GO, G/GO,
T/GO, AP/GO, and C/GO mismatches than that of the truncated M25. The
largest difference between MutY and M25 is the binding of T/GO- and
A/GO-containing DNA substrates. Because T/GO and C/GO are not catalytic
substrates of MutY (Fig. 1), the tight binding of these substrates by
MutY through its C-terminal domain is surprising. When an AP site was
placed opposite G or GO, M25 displayed similar binding affinity to
AP/G, but weaker binding affinity to AP/GO relative to the intact
enzyme. M25 showed lower binding affinity to DNA substrates containing
GO paired with any partner mismatches than intact MutY. Thus, the
C-terminal domain of MutY contributes to the high binding affinity of
MutY to GO.
Catalytic Activities of M25--
In the trapping assay in the
presence of sodium borohydride, expressed M25 protein produced about
2-fold less covalent protein-DNA complex on A/G-containing DNA than
intact MutY at the protein/DNA ratios greater than 10 (Fig.
2A, lanes 1-3 and
6-8). At lower protein/DNA ratios, a 2-fold amount of M25
was needed to produce the same amount of complex as MutY. With A/GO
mismatch, when M25 was present in large enzyme excess (Fig.
2B, lane 6), the extent of protein-DNA covalent
complex formation was 2-fold less than that of intact MutY (Fig.
2B, lanes 1-4). However, the amount of M25
needed to produce the same amount of covalent complex was 40-fold
higher than that of intact MutY with A/GO-containing DNA (Fig.
2B, compare lane 5 with lane 9).
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Fig. 2.
Formation of covalent complexes of MutY and
M25 with A/G- and A/GO-containing DNA in the presence of
NaBH4. Oligonucleotide substrates (1.8 fmol) were
incubated with various amounts of MutY (lanes 1-5) or M25
(lanes 6-10) in the presence of 0.1 M
NaBH4 at 37 °C for 30 min. A, trapping
reactions with A/G-containing DNA. Lanes 1-5 show results
from reactions containing decreasing amounts of MutY (144, 36, 9, 1.8, 0.45 fmol from lane 1 to lane 5, respectively).
Lanes 6-10 show results from reactions containing
decreasing amounts of M25 (288, 72, 18, 3.6, and 0.9 fmol from
lane 6 to lane 10, respectively). B,
trapping reactions with A/GO-containing DNA. Lanes 1-5 show
results from reactions containing decreasing amounts of MutY (36, 9, 1.8, 0.45, and 0.09 fmol from lane 1 to lane 5,
respectively). Lanes 6-10 show results from reactions
containing decreasing amounts of M25 (288, 72, 18, 3.6, and 0.9 fmol
from lane 6 to lane 10, respectively). Reactions
were stopped by adding 5 µl of 3× dye (9 mM Tris-HCl, pH
6.8, 15% glycerol, 3% SDS, 5% -mercaptoethanol, and 0.3 mg/ml
bromphenol blue) and the products, after heating at 90 °C for 2 min,
were electrophoresed on a 12% SDS-PAGE gel. The positions of free DNA
(Free) and covalent complexes (C-M25 and
C-MutY) are indicated.
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Because of the slow turnover rate of MutY (30, 53), the steady-state
kinetics of the MutY reaction, as measured at 37 °C for 30 min (33),
may not reflect the true reactivity. Thus, we used single-turnover
glycosylase kinetics to compare the reactivities of MutY and M25. Time
course studies to determine the extent of glycosylase activity on both
A/G and A/GO substrates were performed with intact MutY and M25. As
shown in Fig. 3A, the rate of
cleavage of A/G-containing DNA is similar for both intact MutY
(circles) and M25 (diamonds). The times required
to reach 50% of Vmax on A/G-containing DNA are
2.1 and 2.7 min for MutY and M25, respectively. However, the rate of
cleavage of M25 on A/GO-containing DNA is much slower than that of MutY
(Fig. 3B). The times required to reach 50% of
Vmax on A/GO-containing DNA are <0.1 and 1.9 min for MutY and M25, respectively. There is a greater than 20-fold difference in reaction rate. The rate of M25 cleavage of
A/GO-containing DNA is similar to the rates of MutY and M25 cleavage of
A/G-containing DNA (compare diamonds of Fig. 3B
to both curves in Fig. 3A). Thus, M25 has reduced rate of
glycosylase activity on A/GO-containing DNA, compared with intact
MutY.
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Fig. 3.
Time course studies of MutY and M25
glycosylase activities. A/G (panel A) and A/GO
(panel B) containing 20-mer oligonucleotides (1.8 fmol) were
incubated at 37 °C with 72 fmol of MutY (circles) or 72 fmol of M25 (diamonds) for various times. The products,
after reaction, were treated with piperidine, lyophilized, resuspended
in formamide dye, heated at 90 °C for 2 min, and analyzed on a 14%
denaturing sequencing gel. Data were from PhosphorImager quantitative
analyses of gel images over three experiments. Percentages of DNA
cleaved were plotted as a function of time.
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The results of Fig. 3 also showed that there was no difference between
MutY and M25 when the reactions proceeded for more than 15 min with
both A/G and A/GO mismatches. At the steady-state, MutY glycosylase
activity with A/GO mismatch is weaker than with A/G-containing DNA.
This result is consistent with the previous finding (33).
Inhibition of MutM (Fpg) Activity by MutY and M25--
DNA
substrates containing C/GO and T/GO are catalytic substrates of MutM
(Fpg) (54) but not of MutY (Fig. 1) although they are bound tightly by
MutY (Table I). To explore the significance of these properties, we
tried to include MutY in the MutM glycosylase reaction. Both DNA
substrates were labeled at the 5' ends of GO-containing strands and
preincubated with increasing amounts of MutY before MutM was added. As
shown in Fig. 4, A and
B, MutY could inhibit MutM activity on both C/GO and T/GO
mismatches. MutM activity was reduced to 50% at MutY/MutM ratios of 32 and 0.6 for C/GO and T/GO, respectively (Table
II). Because M25 has lower affinities to
C/GO and T/GO than does the intact MutY (Table I), it requires a higher
amount of enzyme compared with MutY to achieve the same extent of
inhibition on MutM activity (Fig. 4, A and B,
compare circles and diamonds). MutM activity was
reduced to 50% at M25/MutM ratios of 1600 and 80 for C/GO and T/GO
mismatches, respectively (Table II). The extent of MutM activity
inhibition by MutY and M25 is consistent with the Kd
values of these proteins with these substrates.
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Fig. 4.
Inhibition of MutM activity by MutY and
M25. Oligonucleotides (labeled at 5'-ends of the GO-strands, 1.8 fmol) containing a C/GO (panel A), T/GO (panel
B), G/GO (panel C), or AP/GO (panel D)
mismatches were preincubated for 2 min at room temperature with various
amounts of MutY, M25, or diluent. MutM was added afterward to a final
concentration of 0.09 nM for C/GO, T/GO, and G/GO
mismatches and 0.36 nM for AP/GO mismatch, and the reaction
was further incubated at 37 °C for 28 min. The products, after
reaction were lyophilized (without piperidine treatment), resuspended
in formamide dye, heated at 90 °C for 2 min, and analyzed on a 14%
denaturing sequencing gel. Data were from PhosphorImager quantitative
analyses of gel images from more than three experiments. Percentages of
MutM activity relative to without MutY sample were plotted as a
function of MutY (circles) or M25 (diamonds) to
MutM molar ratios.
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DNA with a G/GO mismatch is a weak substrate of MutY (Fig. 1) but is
cleaved very well by MutM (54). When MutY and M25 were added to the
MutM reaction with G/GO-containing DNA, strong inhibition was observed
(Fig. 4C). MutM activity on G/GO mismatch was reduced to
50% at MutY/MutM and M25/MutM ratio of 0.7 and 6, respectively (Table
II). The extent of MutM activity inhibition by MutY and M25 is in
agreement with the Kd values of these proteins with
the substrate, however, M25 appears to exhibit greater inhibition of
MutM activity than as predicted from its Kd value.
AP/GO-containing DNA is a substrate for both MutY and MutM. The AP
lyase activity of MutY can cleave at the 3' side of the AP site (37,
45). MutM can excise the GO base and cleave the AP sites on both DNA
strands leading to a double-strand break (54). As shown in Fig.
4D, MutY could block MutM cleavage on the GO-strand of AP/GO
mismatch. M25, which had an 18-fold lower binding affinity to AP/GO
than the intact MutY, inhibited MutM activity on AP/GO to a lesser
degree than MutY (Fig. 4D, compare circles and
diamonds). MutM activity on AP/GO-containing DNA was reduced
to 50% by MutY and M25 at enzyme/MutM ratios of 0.3 and 16, respectively (Table II).
Deletion of C-terminal Domain of MutY Causes a Mutator
Phenotype--
The micA (mutY) mutant PR68 was originally
isolated in a mutL Su background and was
defective in a repair pathway that removes the adenines from A/G
mismatches (5). The mutY mutant strain PR70 (same as PR68
but MutL+) also displays a mutator phenotype (5).
The mutY gene in PR70 is interrupted by a mini
Tn10Kan (5) at a site about 750 base pairs downstream from
the AUG start codon (22). To determine the exact insertion site of the
transposon on this mutant allele, we sequenced the clone, pJTW1-1 (22), bearing micA68 with MutY its own promoter in plasmid pBR322. As shown
in Fig. 5A, mini
Tn10Kan interrupts at nucleotide 747 of mutY gene
followed by a stop codon. PR70 cells should express a 249-residue
polypeptide from the N terminus of MutY. As expected, in a Western blot
with MutY antibodies, a band at approximately 27 kDa was detected in
PR70 and PR70 containing plasmid pJTW1-1 (Fig. 5B, lanes 2 and 3). The 249-residue polypeptide may be less stable or
less reactive to the MutY antibodies than the intact MutY as it is
hardly detected in the PR70 extract and the truncated protein is
present at lower levels in PR70/pJTW1-1 than MutY in PR8 (wild-type) as
detected by Western blotting.
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Fig. 5.
Characterization of micA68
mutation. A, map of micA68. DNA sequencing of
plasmid pJTW1-1 (22) bearing the micA68 mutant
mutY gene revealed the mini Tn10 insertion site
at nucleotide 747 of the mutY gene and a stop codon
immediately after codon 249. A 249-residue polypeptide from the N
terminus of MutY was expected to be produced from the interrupted
mutY gene. B, Western blotting with MutY
antibodies. Extract of PR8 (80 µg) expressed the intact 39-kDa MutY
protein (lane 1). A band about 27 kDa was detected in cell
extracts (80 µg) from PR70 (lane 2) and PR70 containing
pJTW1-1 (lane 3). Purified M25 and MutY proteins were
included as markers in lanes 4 and 5,
respectively. C, gel shifting assay. A/G- and
A/GO-containing 20-mer oligonucleotide was assayed for binding with 10 µg (odd lanes) or 5 µg (even lanes) of cell
extracts at 37 °C for 30 min. Lanes 1 and 2,
PR8 with A/G-containing DNA; lanes 3 and 4, PR70
with A/G-containing DNA; lanes 5 and 6,
PR70/pJTW1-1 with A/G-containing DNA; lanes 7 and
8, PR8 with A/GO-containing DNA; lanes 9 and
10, PR70 with A/GO-containing DNA; lanes 11 and
12, PR70/pJTW1-1 with A/GO-containing DNA. The binding
products were analyzed on an 8% native gel in 50 mM Tris
borate, pH 8.3, and 1 mM EDTA. Arrows indicate
the positions of MutY·DNA complex (B-MutY), micA68·DNA
complex (B-micA68), and free DNA (Free).
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The extracts of PR68 and PR70 have been shown to be defective in the
methylation-independent A/G mismatch repair on phage f1 heteroduplexes
and A/G-specific cleavage activity using a 120-mer oligonucleotide
substrate (22). When the PR70 extract was re-examined with A/G and A/GO
20-mer DNA substrates, no binding activity could be detected (Fig.
5C, lanes 3-4 and 9-10). Next, we examined MutY binding activity in extracts of PR70 bearing the same mutant allele in
plasmid pJTW1-1. Extracts of PR70 containing pJTW1-1 had stronger binding activity with A/G-containing DNA than did PR8 extracts (Fig.
5C, compare lanes 1 and 2 with
lanes 5 and 6) although MutY in the PR8 extract
was present at higher levels than the truncated protein in the extract
of PR70/pJTW1-1 as detected by Western blotting (Fig. 5B, lanes
1 and 3). However, the PR8 extract had much greater
binding activity with A/GO-containing DNA then did PR70/pJTW1-1 extract
(Fig. 5C, compare lanes 7 and 8 with
lanes 11 and 12). Intact MutY in the PR8 extract
had much tighter binding with A/GO- than to A/G-containing DNA,
however, the truncated protein in the PR70 extract had similar but weak
binding with both A/GO and A/G mismatches. Thus, the truncated
249-residue MutY polypeptide encoded by the micA68, as with
M25, has lower binding affinity to GO than intact MutY.
The mutation frequency of PR70 has been determined to be more than
40-fold higher than the wild-type cells as measured by the incidence of
rifampicin-resistant revertants (5, 55). As shown in Table
III, the mutation frequencies of PR70 and
PR70/pJTW1-1 was 75- and 32-fold higher than the wild-type cells,
respectively. Although the mutation frequency of PR70/pJTW1-1 is not as
high as PR70, it displays a mutator phenotype. The phenotype and the biochemical properties of the encoded protein by the micA68
allele strongly support that deletion of the GO-specific C-terminal
domain of MutY could lead to a mutator phenotype. It also suggests that repair of A/GO mismatches is the primary role of MutY pathway.
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DISCUSSION |
MutY has been identified as an adenine glycosylase that repairs
A/G, A/C, and A/GO mismatches (3, 4, 7, 35). Here, we showed that MutY
is also a guanine glycosylase repairing G/GO-containing DNA. The
binding affinity of MutY with G/GO is slightly lower than that with
A/GO-containing DNA but higher than that with A/G-containing DNA.
However, the catalytic activity of MutY to G/GO-containing DNA is much
lower than that to A/G- and A/GO-containing substrates. The adenine
specificity to A/G and A/GO is consistent with the mutation phenotype
of mutY mutants for G:C to T:A transversions (3, 5, 56). The
weak in vitro A/C and G/GO repair activities of MutY may
remove the misincorporated A and G from C and GO on the template
strands, respectively. Thus, one may predict that mutY
mutants have higher mutation frequencies for C:G to T:A and G:C to C:G.
Nghiem et al. (56) and Radicella et al. (5) found that the mutY mutants had higher mutation rates for G:C to
T:A transversions but not other types of mutations compared with the wild-type cells. It is possible that A/C and G/GO mismatches are repaired by other pathways such as the MutHLS system. Recently, Zhang
et al. (57) found that an E. coli mutY mutant
also has an increased mutation rate for G:C to C:G transversions and
that MutY has guanine glycosylase activity on G/GO but not G/G
mismatches. Their experimental design is similar to that of Nghiem
et al. (56) except that the E. coli cells were
incubated on glucose minimal media for 10 days instead of 3 days. The
delayed mutation accumulation of G:C to C:G transversions may be due to
the inefficiency of MutY repair to G/GO mismatches or miscoding of an
oxidized form of GO that appeared at the stationary phase (58).
Full-length MutY binds to A/GO-containing DNA 80-fold better than it
does to A/G-containing DNA (20). We have shown that the specificity for
A/GO binding can be ascribed mainly to the C-terminal domain of MutY,
the non-endo III like domain (39). Here, we showed that M25 and MutY
have similar binding affinity with an A/G mismatch, however, M25 has
much lower affinity to DNA substrates containing GO when paired with
any partner mismatches than intact MutY. Thus, the high binding
affinity of MutY to GO-containing DNA is contributed mainly by its
C-terminal domain. Because MutY has different catalytic activities
(Fig. 1) and low turnover rate (30, 53) with these mismatches, the
Kd measurement for these substrates may not reflect
true equilibrium values. In the case of A/G, A/GO, and A/C, MutY may
bind to either the base-base mismatch substrates or the processed
products such as AP site or cleaved site. The specific GO-binding role
of C-terminal domain of MutY was also described by Noll et
al. (47) but not found by Manuel and Lloyd (37). The N-terminal
domain (p26) studied by Manuel and Lloyd (37) consists of residues
1-225 which is 1 amino acid shorter than M25 used in this study as
well as by Noll et al. (47) and Gogos et al.
(39). However, the functional difference between M25 and P26 is
probably not derived from 1 amino acid difference.
M25 also has weaker trapping and slower glycosylase activities than
MutY with A/GO-containing DNA. When M25 is present in large enzyme
excess, the extent of protein-DNA covalent complex formation is about
2-fold less than that of intact MutY (Fig. 2). However, a 40-fold
higher amount of M25 is needed to produce the same amount of covalent
complex as that of intact MutY with A/GO-containing DNA. At the
steady-state, there was no difference in glycosylase activity between
MutY and M25 when the reactions proceeded for more than 15 min for both
A/G and A/GO. At the pre-steady-state, M25 and MutY have similar rates
of glycosylase activity with an A/G mismatch, however, M25 reacts much
slower than MutY to an A/GO mismatch. Thus, both the high binding
affinity and fast catalytic activity of MutY to GO-containing DNA are
contributed mainly by its C-terminal domain. Furthermore, an E. coli mutY strain that produces an N-terminal 249-residue truncated
MutY confers a mutator phenotype. The C-terminal mutY
deletion allele encodes a polypeptide whose activity is similar to M25.
These findings strongly support that the C-terminal domain of MutY is
critical for mutation avoidance of oxidative damage. Thus, in
vivo, the A/GO mismatches may be the most important substrates of
MutY because both prokaryotic and eukaryotic DNA polymerases misinsert
A opposite GO at high frequencies (9, 15, 16, 58).
M25, spanning the region homologous with E. coli endo III,
is the catalytic domain of MutY (cdMutY) (37, 39, 40). Several DNA
binding motifs have been identified in the x-ray crystal structure: a
helix-hairpin-helix, a pseudo-helix-hairpin-helix, a positively charged
groove with the adenine binding pocket, and an iron-sulfur cluster loop
(24). These motifs are responsible for interaction with the DNA
phosphates and the mismatched adenine. The crystal structure of the
cdMutY also suggests some candidate residues for the recognition of the
base opposite the adenine. These include the 
2-3 motif,
particularly the conserved Gln41, for the specific
recognition of G or GO at a syn configuration through the
DNA minor groove. Our results show that the major determinants of the
mismatched GO reside in the C-terminal domain of MutY. We propose that
the DNA is embedded between the catalytic and C-terminal domains of
MutY. In this model, the C-terminal domain functions like a clamp to
hold the GO-containing strand. This C-terminal clamp is more open with
an A/G mismatch than with an A/GO mismatch. The contacts of MutY with
the GO-containing strand may be important for promoting base-flipping
of the mismatched A. The residues of the C-terminal domain of MutY
involved in this GO specificity remain undetermined. It would be
valuable to have the detailed structure of the intact MutY·DNA complex.
Noll et al. (47) pointed out an interesting sequence
homology between the C-terminal domain of MutY and MutT. The MutT
protein (14 kDa in size) hydrolyzes nucleoside triphosphates, with
8-oxo-dGTP as the best substrate, to nucleoside monophosphates and
pyrophosphates (10-12). Both polypeptides are similar in size and can
recognize the GO nucleotide. However, the C-terminal domain of MutY by
itself has no detectable function (data not shown), unlike MutT. Thus, the C-terminal domain of MutY has evolved to function differently from
MutT and it may enhance the N-terminal domain to interact with the
phosphate backbone and GO base in DNA.
The tight binding of MutY to T/GO, G/GO, and AP/GO may have biological
significance. MutM is able to remove GO from T/GO, G/GO, AP/GO, and
C/GO efficiently in vitro (54). It is possible that MutY
continues to bind AP/GO mismatches after its glycosylase action in
order to prevent removal of GO or cleavage at the AP site by MutM
before MutY initiated repair is complete. The inhibition of MutM
activity is especially important if T/GO and G/GO mismatches arise from
misincorporation of T and G opposite oxidized template guanines as well
as if T/GO mismatches are derived from deamination of 5-methylcytosine
opposite GO. DNA polymerases may incorporate G or T opposite the GO
lesions as suggested by Braun et al. (59). An oxidized form
of GO, possibly guanidinohydantoin, may direct misincorporation of
dNTPs during DNA synthesis (58). The removal of GO from T/GO and G/GO
mismatches when GO is on the parental strands will lead to G:C to A:T
transitions and G:C to C:G transversions. Hence, it is reasonable that
MutM activity on these unfavorable mismatches is inhibited before other
repair pathways remove the base opposite GO. The possible pathways
employed to repair these unfavorable mismatches are the long-patch
MutHLS mismatch repair and vsr-dependent very
short-patch repair (60). Recently, yeast MSH2/MSH6 heterodimer
(E. coli MutS homologs) has been shown to bind A/GO
mismatches and be involved in GO repair (61). Thus, it is possible that
MutS homologs may be involved in repair of T/GO and G/GO mismatches.
Our data in Fig. 4 indicate that the MutY protein is involved in this
regulation of MutM activity through its C-terminal domain. It has been
suggested by Bridges et al. (62) that MutY may regulate MutM
activity in resting cells.
Our model for MutY attenuation of MutM activity is shown in Fig.
6. When MutY binds A/G and A/GO, the
adenine is flipped out of the DNA helix into the binding pocket and the
MutY glycosylase then excises the base (Fig. 6A). When MutY
binds T/GO, the T base is not flipped out, no repair occurs, and MutY
blocks the MutM binding to this substrate (Fig. 6B). When
MutY encounters a G/GO mismatch, the base flipping and repair are very
slow; MutY can block the MutM binding when it remains bound to this
substrate (Fig. 6C). This additional role of MutY in
blocking MutM activity is applicable only when GO is on the parental
strands. It is possible that MutY is orientated at the replication fork
with the N-terminal domain on the daughter strand and C-terminal domain
on the parental strand by an unknown mechanism. With this physical
polarity, MutY may repair the misincorporated A opposite template GO
but not repair template A opposite misinserted GO.
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Fig. 6.
Model for MutY blockage of MutM
activity. DNA strands at the replication fork are represented as
lines. The N-terminal and C-terminal domains of MutY are
shown in large and small oval-shaped circles,
respectively. The gray-filled multi-pointed oval inside the
large oval marks the adenine binding pocket. A,
MutY binding with A/GO mismatch. When MutY binds A/GO or A/G, the
adenine is flipped out of the DNA helix into the binding pocket and the
MutY glycosylase then excises the base for repair. B, MutY
binding with T/GO mismatch. When MutY binds T/GO, the T base is not
flipped out, no repair occurs but MutY blocks the MutM binding to this
substrate. C, MutY binding with G/GO mismatch. When MutY
encounters a G/GO mismatch, the base flipping and repair are very slow,
MutY can block MutM binding because it remains bound to this
substrate.
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TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 410-706-4356;
Fax: 410-706-1787; E-mail: aluchang@umaryland.edu.
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