(Received for publication, February 28, 1995; and in revised form, July 19, 1995)
From the
Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro-8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein.
Multiple mismatch repair pathways with different mispair
specificities and different size repair tracts are utilized by Escherichia coli to reduce replicative errors and to protect
its DNA from various types of damage(1) . One of the
short-patch repair pathways requires mutY gene function and is
independent of dam-methylation(2, 3, 4) . E.
coli mutY (or micA) mutants have higher mutation rates
for CG to A
T transversions(3, 5) . The
MutY pathway specifically repairs A/G mismatches to C/G base pairs (2, 3, 4, 6) and repairs A/C to G/C
at a much lower rate(2, 3, 7) . MutY can also
act on adenines mispaired with 7,8-dihydro-8-oxo-guanine (GO) (
)or 7,8-dihydro-8-oxo-adenine (AO)(8, 9) .
The GO lesion is one of the most stable products of oxidative damage to
DNA known. A role for the MutY pathway in E. coli is to remove
misincorporated adenines opposite G or GO following DNA
replication(8, 10) . Adenines are frequently
incorporated opposite GO bases during DNA replication in vitro(11) and in vivo(12) . A second round of
replication through this mismatch subsequently leads to a C
G to
A
T
transversion(12, 13, 14, 15) . E. coli uses MutY, MutM, and MutT to defend against the
mutagenic effects of GO lesions(10, 16) . The MutT
protein eliminates 8-oxo-dGTP from the nucleotide pool by its
nucleoside triphosphatase
activity(17, 18, 19) . The MutM protein (FPG
protein) provides a second level of defense by removing both
ring-opened purine lesions and mutagenic GO
adducts(20, 21) . MutM removes GO lesions efficiently
from C/GO but poorly from A/GO(21) . MutY works at a third
level by correcting replicative errors that result from
misincorporation of A opposite GO(8, 9) .
The 39-kDa MutY protein is an iron-sulfur protein, which has homology with E. coli endonuclease III(7, 22, 23) . The MutY protein was shown by Tsai-Wu et al.(7) to have both DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities, although the AP endonuclease activity could not be detected by Au et al.(24) in their MutY preparation. The DNA glycosylase activity removes the adenine bases from the A/G, A/C, A/GO, and A/AO mismatches(7, 9, 24) , and the AP endonuclease activity cleaves the first phosphodiester bond 3` to the AP site(7, 25) .
The activity of MutY on its mismatched DNA substrates is influenced by the neighboring sequence composition(2, 3, 25) . Structural studies have demonstrated that A/G mispairs can adopt three possible configurations (A(anti)-G(anti), A(anti)-G(syn), and A(syn)-G(anti)), depending on their neighboring sequences(26, 27, 28, 29, 30) . It is unclear which configuration of A/G mispair is recognized by MutY. Based on the common features of MutY substrates (A/G, A/GO, A/C, and A/AO), it has been suggested that the N-1 of adenine may be protonated and/or that the guanine is in the syn configuration(7, 9) . In the work described here, we have explored the potential purine and phosphate contacts involved in MutY recognition. Alkylation interference experiments have demonstrated that MutY specifically interacts with mismatched A and G as well as neighboring sequences. This may explain the sequence effect on the repair efficiency of MutY. Defined oligonucleotides containing various purines were used in this study to examine the substrate specificity of MutY protein and to establish the role of mispair functional groups in MutY recognition and catalysis. Our results add nebularine/G (N/G), inosine/G (I/G), and 2-amino-purine (2AP/G) to the growing list of recognized substrates of MutY, although I/G and 2AP/G are not cleaved well by MutY.
Figure 1: Quantitation of ethylation interference effects. The 40-mer heteroduplex DNA containing an A/G mismatch (boxed) was 5`-end-labeled on the A-strand (toppanel) or G-strand (bottompanel). The DNA was partially ethylated with nitrosourea, purified, and bound to MutY. After binding to MutY, free and bound DNA were separated by 4% native polyacrylamide gel electrophoresis, eluted, and chemically cleaved(37) . Control samples are 40-mer heteroduplex DNA treated by the same procedures without adding MutY protein. Cleavage products were then analyzed on 10% DNA sequencing gels and exposed to x-ray films. PanelB shows a representative autoradiogram of ethylation interference on the G-strand. Results shown in panelA are based on densitometric analysis of these autoradiograms. The extent of interference is expressed as the ratio of the peak area of each band derived from free DNA in MutY-added samples and from control samples without MutY. The experiments were performed in quadruplicate, and the results were averaged. Errorbars are ±1 S.D. No significant interference was observed for those regions of the 40-base pair DNA, which are not shown.
Figure 2: Quantitation of methylation interference effects. The 40-mer heteroduplex DNA containing an A/G mismatch (boxed) was 5`-end labeled on the A-strand (toppanel) or G-strand (bottompanel). The DNA was partially methylated with dimethylsulfate, purified, and bound to MutY. After binding to MutY, free and bound DNA were separated by 4% native polyacrylamide gel electrophoresis, eluted, and chemically cleaved by the A > G reaction(33) . Control samples are 40-mer heteroduplex DNA treated by the same procedures without adding MutY protein. Cleavage products were then analyzed on 10% DNA sequencing gels and exposed to x-ray films. PanelB shows a representative autoradiogram of methylation interference on the G-strand. Interference was quantitated and shown in panelA as described in Fig. 1. The data shown here are the average of at least five experiments. Errorbars are ±1 S.D.
Fig. 3summarizes the distribution of potential purine and phosphate contacts of MutY as deduced from the alkylation interference experiments. MutY covers about 12 base pairs of the A/G-containing DNA and has more contacts on the G-strand.
Figure 3: Summary of the alkylation interference on specific complexes between MutY protein and DNA containing an A/G mismatch. The methylation interference sites are indicated by circles, and the ethylation interference sites are marked by triangles. The sequence positions are labeled with respect to the A-strand from the 5`-end of the 40-mer DNA. The A/G mismatch is boxed, and the phosphodiester bond cleaved by MutY AP endonuclease is indicated by an arrow.
Figure 4: Purine base analogs.
Figure 5: Effect of single inosine and uracil substitutions on MutY endonuclease activity. DNA substrates (3`-end-labeled 20-mer, 1.8 fmol) were assayed for the endonuclease activity at 37 °C for 30 min with 2 nM MutY. The cleaved DNA fragment (N) and intact DNA (I) are indicated by arrows. A minor band above the major DNA species is an impurity. Oligonucleotides used are A/G-20 (lane1), A/G-I1 (lane2), A/G-U1 (lane3), A/G-U2 (lane4), A/I-20 (lane5), A/G-I2 (lane6), and A/G-U3 (lane7) (see Table 1for sequence).
Figure 6: Cleavage of mismatch-containing oligonucleotides by MutY protein. DNA substrates (3`-end-labeled 20-mer, 1.8 fmol) were assayed for endonuclease activity at 37 °C for 30 min with 168 nM MutY (note that this concentration is higher than that used in the experiments of Fig. 5). The cleaved DNA fragment (N) and intact DNA (I) are indicated by arrows. In some DNA samples, two bands of DNA differing by one nucleotide are present. The exact cause is not clear, but these DNA were labeled as described under ``Experimental Procedures,'' and then four cold nucleotide triphosphates were added for an additional 5-min incubation period. Oligonucleotides containing the following mismatches or homoduplexes were used: A/G (lane1), A/C (lane2), A/GO (A/O, lane3), N/G (lane4), I/G (lane5), 2AP/G (2/G, lane6), Z/G (lane7), A/I (lane8), A/2AP (A/2, lane9), C/G (lane10), and A/T (lane11) (see Table 1for sequence). Endonuclease activity is defined as that resulting in cleavage of 0.018 fmol (1%) of labeled DNA in 30 min at 37 °C.
Figure 7: Binding of mismatch-containing oligonucleotides by MutY protein. DNA substrates (3`-end-labeled 20-mer, 1.8 fmol) were assayed for the binding at 37 °C for 30 min with 27 nM MutY. Oligonucleotides used were the same as in Fig. 6. The protein-bound and free DNA are indicated by arrows. Under this condition, some cleaved product of A/G- and N/G-containing DNA were dissociated from MutY and migrated off the gel (see Fig. 8A). Note that little free DNA was present in lanes1-6 at this MutY concentration and complexes migrated at slightly different rates.
Figure 8:
Apparent dissociation constant (K) determination of MutY protein binding
to a 20-mer oligonucleotide containing a single A/G or A/I mismatch. A, 1.8 fmol of 3`-end-labeled A/G-20 was incubated with
increasing amounts of MutY (0, 0.42, 0.84, 1.68, 3.36, 6.72, 13.4,
26.9, and 53.7 nM; lanes1-9,
respectively) in 20 µl of reaction mixture at 37 °C for 30 min.
Binding products were analyzed on an 8% polyacrylamide gel followed by
autoradiography. The protein-bound, free, and nicked DNA are indicated.
The DNA band indicated by an openarrow migrated at
the same position of single-stranded DNA and appeared not to be a
substrate for MutY. B, reactions were performed as described
in A except A/I-containing DNA was used as substrate with
increasing amounts of MutY (0, 26.9, 53.7, 107, 168, 336, 671, 1340,
and 2690 nM; lanes1-9, respectively).
Multiple bound bands (including one at the lane origin) were observed
and were all included as the bound fraction. An openarrow indicates the position of single-stranded DNA. There were no
detectable nicked products. C and D, MutY binding
reactions to A/G- and A/I-containing DNA, respectively, were performed
in triplicate. The bands corresponding to the bound and free fractions
of DNA (as in A and B) were excised and quantified by
liquid scintillation counting. The % of bound A/G-containing DNA was
determined, plotted versus MutY concentration, and the value
for K
was determined by
Enzfitter(42) . The apparent K
for A/I-containing DNA was determined as the concentration
of MutY that results in 50% binding of input DNA. In this case, MutY
binding to A/I-20 is not saturated at the highest MutY concentration
tested and is not suitable for Enzfitter
analysis.
The apparent
dissociation constants (K) of MutY from the
different mismatches were determined. Representative autoradiograms of
the binding assay and the corresponding binding curve for MutY to A/G-
and A/I-containing 20-mer DNA are shown in Fig. 8(panels
A-D). In A/G binding assays, there was a band that migrated
faster than the free DNA and represented about 5 and 15% of input DNA
at 3.4 and 53.7 nM MutY, respectively, indicating that some
A/G-containing DNA was cleaved by and dissociated from MutY (Fig. 8A). Cleavage products were not used in the K
calculation because their incusion would
negligibly and improperly increase the apparent values. The cleaved
free DNA band in native gel was not observed in the binding of MutY at
53.7 nM to A/GO-containing DNA (data not shown). Some slower
mobility complexes (either polymers or aggregated forms) were observed
in binding assays with low affinity DNA substrates (A/2AP, Z/G, A/I,
A/T, and C/G) when the concentration of MutY protein was higher than
0.34 µM (Fig. 8B). The results of these
experiments, performed in triplicate, are summarized in Table 2.
The apparent K values for 20-mer and 44-mer DNA
containing an A/G mismatch were 5.3 and 1.8 nM, respectively.
MutY bound strongly to the 20-mer duplex with an A/GO mismatch
(apparent K
= 66 pM). This binding
is 80-fold greater than that to A/G-containing DNA. The binding
affinities of MutY for 20-mer oligonucleotides containing N/G and I/G
were comparable to that for A/G. MutY bound weakly to duplex DNA
containing A/C and 2-AP/G, much weaker to DNA with A/2AP, Z/G, and A/I,
and very weakly to duplex DNA containing a matched A/T or C/G base
pair. Reproducibly, MutY bound slightly better to A/T- than to
C/G-containing DNA (Fig. 7, lanes10 and 11). The binding affinity of MutY was of the following order:
A/GO A/G = N/G > I/G > 2AP/G > A/C A/2AP > Z/G
> A/I
A/T
C/G.
DNA containing A/G, A/C, A/GO, or A/AO mismatch has been
shown to be the substrate of MutY
protein(7, 9, 24, 25) . The MutY
protein removes the mispaired adenines from the mismatches by
glycosylase activity(7, 24) . Tsai-Wu et al.(7) showed that MutY also has AP endonuclease activity.
However, the AP endonuclease activity could not be detected by Au et al.(24) in their MutY preparation. The reason for
this discrepancy is not clear. The detection of the dissociated nicked
product in a native gel (Fig. 8A) argues that the
endonuclease activity observed in MutY reaction is not caused by
heating at high pH, which may catalyze a -elimination at the AP
site. Therefore, the AP endonuclease (or lyase) activity of MutY
appears intrinsic. DNA phosphate ethylation experiments suggest that
MutY interacts with phosphate residues spanning about 12 nucleotide
pairs encompassing the A/G mismatch (Fig. 3). MutY binds DNA
mainly via five major ionic bonds. Two phosphates with significant
effects upon ethylation are located 5 and 7 phosphodiester bonds 3` and
5` to the mismatched G, respectively. This defines a large component of
electrostatic binding energy involved in the binding of MutY to
phosphates outside of the A/G mismatch. Interestingly, ethylation at
the first phosphodiester bond 3` to the mispaired A that is attacked by
the MutY AP endonuclease did not have a substantial interference effect
on MutY binding (Fig. 1).
Methylation interference experiments with 40-mer DNA containing an A/G mismatch (Table 1) reveal that MutY interacts with purines including the mismatched A and G and two bases on either side of the mismatch (Fig. 3). The N-3 group of A-20 on the G-strand is located in the minor groove while the N-7 groups of G-23 on the A-strand and G-24 on the G-strand are located in the major groove. Substitution of these two guanines by inosines indicates that the 2-amino groups of these guanines, located in the minor groove, are also important in MutY recognition. If substantial helix perturbation is not associated with MutY binding, these findings suggest that MutY binds DNA in both the major and minor grooves in the vicinity of the A/G mismatch. The involvement of purines flanking the mismatch in MutY binding may reflect the effect of neighboring sequences on repair and cleavage efficiencies(2, 25, 38) . Although flanking sequence effects on MutY reactivity have not been explored systematically, our findings provide a rationale for earlier observations.
The locations of the N-7 group of mismatched G and the N-3 group of mismatched A depend on the structure of the A/G mismatch. Structural analyses have shown that A/G can form three possible conformations: A(anti)-G(anti), A(anti)-G(syn), and A(syn)-G(anti), depending on the neighboring environment(26, 27, 28, 29, 30) . Because A/GO is a substrate for MutY protein and it can form a very stable base pair when A is in the anti and GO is in the syn configuration(39) , it is suggested that the A(anti)-G(syn) conformation may be the favored substrate for MutY(9) . The protonated N-1 form of adenine is also suggested for the A/C structure(40) . Although these DNA conformations with bound MutY have yet to be confirmed, we will assume in the following discussion that the A/G-containing substrate for MutY is in the A(anti)-G(syn) conformation, in which both the N-3 group of mismatched A and the N-7 group of mismatched G are located in the minor groove.
The role of functional groups in mismatch recognition and catalysis by MutY protein were further elucidated by binding and endonuclease assays using defined oligonucleotides containing various purine derivatives. The C-6 amino group of mismatched A is not critical for DNA binding or cleavage since duplex DNA containing N/G is bound and cleaved efficiently by MutY. This is a surprising finding because the C-6 amino group of adenine is involved in hydrogen bonding with guanine in all three A/G conformations(26, 30) . The loss of one hydrogen bond would be expected to destabilize the A/G base pairing. Thus, the reactivity of MutY protein cannot be rationalized on the basis of the stability of the mismatched pair alone. However, the presence of a C-6 keto group as in the I/G pair blocks catalysis but not binding by MutY. The C-6 keto group may prevent the use of a protonated N-1 or N-3 as an ``electron sink'' in glycosidic bond cleavage. The replacement of the N-7 group of adenine by carbon as in the Z/G pair results in the loss of both binding and endonuclease activities by MutY. The introduction of a C-2 amino group decreases MutY binding and catalysis (compare 2AP/G with N/G). The C-2 amino and the C-6 keto groups of mismatched G, presented in the major groove as in G(syn)-A(anti), are critical for MutY recognition since A/I and A/2AP mismatches are poorly bound by the enzyme.
When
the C-8 keto group of GO is present as in
GO(syn)-A(anti), the apparent K for MutY decreases by 2 orders of magnitude, yet the endonuclease
activity is reduced 2-fold as compared to the A/G mispair. The presence
of a C-8 keto group in A/GO pair changes the nature of hydrogen bonding
between N-7 of GO and N-1 of A. The N-1 of A is protonated in the
G(syn)-A(anti) pair (27) but is not
protonated in the GO(syn)-A(anti) pair (39) .
These structural differences between A/GO and A/G are located in the
minor groove(39) . The relative binding and catalytic
efficiencies of MutY for A/G- and A/GO-containing DNA substrates may
have biological significance. This may prevent MutM from acting upon
the GO lesions so information on both DNA strands would not get lost.
According to Tchou et al.(41) , the apparent K
values of MutM (FPG) protein for A/GO- and
C/GO-containing DNA were 340 and 8.9 nM, respectively. The
affinity of MutY to A/GO-containing DNA is about 5,000-fold higher than
that of MutM.
When the potential DNA contact sites of MutY elucidated in this study are mapped onto a B-DNA model with an A(anti)-G(syn) mispair, several interesting features are revealed. MutY appears to interact with its DNA substrate on both sides of the double helix as well as in both the major and minor grooves. Most base determinants are accessible from one side of the helix. Contacts on the other side of the helix are mediated mainly through three phosphates (between T-15 and C-16, between T-20 and T-21, and between A-26 and A-27). It is interesting to note that these three phosphates are approximately five base pairs apart and form a line nearly parallel to the helical axis. The information obtained in this study provides a basis for understanding the interaction of MutY with DNA and the molecular mechanisms involved in the recognition of damaged or mismatched bases. Identification of I/G-containing DNA as a recognized but not a catalyzed substrate will facilitate the formation of protein-DNA co-crystal.