BACKGROUND
reactive oxygen species are generated by endogenous processes such as mitochondrial oxidative phosphorylation as well as exogenously following exposure to ionizing radiation and chemicals  <cit> .
oxidative dna damage including strand breaks and oxidative base lesions are specifically repaired by base excision repair pathways  <cit> .
the first step in this pathway is carried out by a lesion-specific dna glycosylase, which cleaves the n-glycosidic bond between the base and deoxyribose sugar  <cit> .
the most abundant and highly mutagenic oxidative dna damage lesion is 8-oxo- <dig> -dihydroguanine  that can form base-pair with adenine or cytosine during dna replication to produce a g:c to t:a transversion  <cit> .
in escherichia coli, mutt, mutm , muty, muts, and nei  are involved in defending against the mutagenic effects of 8-oxog lesions [reviewed in  <cit>  and  <cit> .
the mutt protein has pyrophosphohydrolase activity, which eliminates 8-oxo-dgtp from the nucleotide pool.
mutm glycosylase  removes both mutagenic go adducts and ring-opened purine lesions paired with cytosines.
muts and muty increase replication fidelity by removing the adenines misincorporated opposite go or g  <cit> , and thus reduce g:c to t:a transversions.
nei can excise go, when go is paired opposite a cytosine or adenine and can serve as a backup pathway to repair 8-oxog in the absence of mutm and muty  <cit> .
unlike most dna glycosylases, muty does not target a damaged base, but a normal base, e.g., removing a mispaired with go, c, and 5-hydroxyuracil .
in addition, muty can excise guanines from g/go mismatches  <cit> .
muty also binds tightly to dna containing t/go and c/go, however, it exhibits no catalytic activity on them  <cit> .
the n-terminal domain of ecmuty retains the catalytic activity  <cit>  while the c-terminal domain of muty plays an important role in the recognition of go lesions  <cit> .
the x-ray crystal structures of ecmuty catalytic domain with bound adenine  <cit>  and intact bacillus stearothermophilus muty  bound to dna  <cit>  show that muty distorts the bound dna substrate and the mismatched a is flipped out of the helix.
in the active site pocket of ecmuty, seven amino acids  are involved in adenine binding   <cit> .
v <dig> and q <dig> of ecmuty are proposed to be key residues in determining substrate specificity.
v <dig> is considered to be important for glycosylase activity because v45n is inactive  <cit>  and is conserved among muty family members .
q <dig> forms a hydrogen bond with adenine at the domain interface of ecmuty, but is less conserved  <cit> .
the n-terminal domain of muty shares similar structure, including the helix-hairpin-helix  and gly/pro asp loop motifs , with alka, endoiii, and ogg <dig>  the g/t mismatch glycosylase mig.mthi of methanobacterium thermoautotrophicim is a member of the hhh superfamily  <cit>  and is involved in reducing spontaneous deaminated bases from 5-methyl-cytosine residues.
the substrate specificity of mig.mthi is in the order: u/g = t/g > g/g > t/c = u/c > a/g.
in the binding pocket for the target base, mig.mthi shares six out of seven residues of ecmuty's adenine binding .
the only non-conserved residue in this binding pocket is q <dig> of ecmuty, which corresponds to l <dig> of mig.mthi .
interestingly, fondufe-mittendorf et al.
<cit>  could convert mig.mthi into a muty-like glycosylase with altered substrate preference of mig.mthi from t/g to a/g by replacing two residues  in the substrate binding pocket.
their data point to the potential importance of v <dig> and q <dig> in ecmuty substrate recognition.
in this study, we have investigated the role of v <dig> and q <dig> of ecmuty by targeted mutagenesis.
v45a, q182l, and v45a/q182l mutant proteins have reduced binding affinity and glycosylase activity to dna containing a/g or a/go mismatches.
these mutants are impaired to complement e. coli muty mutants in vivo.
the affinities to t/g dramatically increased in v45a and q182l mutants, but not in the v45a/q182l double mutant.
however, v45a, q182l, and v45a/q182l mutant proteins do not exhibit glycosylase activity towards t/g or t/go.
therefore, v <dig> and q <dig> of ecmuty are important for the adenine-specific activity of ecmuty.
RESULTS
selection of ecmuty mutation
the study by fondufe-mittendorf et al.
<cit>  indicates that the substrate preference of mig.mthi can be altered from t/g to a/g by replacing two residues  in the substrate binding pocket.
their results are in agreement that v <dig> and q <dig> of ecmuty are the key determinants of adenine specificity  <cit> .
according to the adenine soaked ecmuty structure   <cit> , v <dig> is located in the minor-groove reading α2–α <dig> motif  and q <dig> is located in the adenine recognition α <dig> motif .
interestingly, the proximity of v <dig> and q <dig> to adenine in ecmuty structure closely correlates with the bsmuty structure with the dna product and cleaved adenine  <cit> .
however, the residues corresponding to val <dig> and gln <dig> in d144n bsmuty with a/go-containing dna   <cit>  do not directly make contacts with the adenine of the a/go mismatch.
in the later structure, the val seems to be positioned below the adenine, but the glu <dig> of bsmuty  is far away from adenine.
the fact that there are differences in adenine recognition among various muty structures raises a question whether these residues really are involved directly in adenine recognition.
to determine whether these two residues are important for adenine recognition and excision, we carried out targeted mutagenesis study to determine the effect of ecmuty v45a, q182l, and double mutant on repair of a/g- and a/go-containing dna.
we also further determined whether their substrate specificity is altered in terms of binding or catalysis of t/g or t/go mismatches.
a/g and a/go binding and glycosylase activity
we compared the binding affinities of v45a, q182l, and v45a/q182l double mutants with wild-type ecmuty to dna substrates containing a/g and a/go mismatches.
first, we determined the active site concentrations of these proteins by performing a/go binding with dna concentrations which are at least 17-fold higher than their estimated dissociation constants .
figure  <dig> shows the average results from two independent experiments with  <dig> nm of a/go-dna.
the increase in wild-type  ecmuty binding reached a maximum at a concentration about  <dig> nm of muty and  <dig>  nm dna was bound .
this horizontal line intercepts with the slope line of initial rate at  <dig> nm of muty.
thus, the wt ecmuty preparation is 68% active.
similar analyses showed that v45a, q182l, and v45a/q182l were 48%, 25%, and 40% active, respectively.
dissociation constant value for each mutant was determined with nine enzyme concentrations by at least three independent experiments .
because muty displays a high affinity for its products  <cit> , the kd values measure its affinities to a mixture of the substrate and the product.
compared to wild-type ecmuty of which the kd to a/g is  <dig>  ±  <dig>  nm, the v45a and q182l mutants had kd values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  nm, respectively.
the kd value  of v45a/q182l double mutant was higher than the single mutants.
thus, the affinities to a/g of v45a, q182l, and v45a/q182l are weakened by 5-, 7-, and 12-fold, respectively.
for a/go binding, v45a and q182l mutations resulted in a 94- and 11-fold increase in kd values, respectively.
the v45a/q182l double mutation resulted in a 102-fold increase in kd value .
thus, v45a is more defective in a/go binding than a/g binding as compared to wild-type muty.
it is interesting to note that the double mutant exhibited an additive reduction in binding to both the a/g and a/go as for the single mutants.
these results confirm that v <dig> and q <dig> play important but independent roles in adenine binding.
a kd values  are mean ± standard deviation for more than three experiments using nine protein concentrations.
bthe numbers in parenthesis are the folds of binding affinity relative to wild-type  muty.
because v45a and q182l ecmuty mutants showed weakened a/g and a/go affinities, we examined their glycosylase activities on a/g and a/go .
v45a mutant showed decrease in a/g glycosylase activity at  <dig>  and  <dig>  nm concentrations  while q182l mutant showed no activity at  <dig>  nm and decrease in activity at  <dig> , and  <dig>  nm , as compared to wild-type muty.
the glycosylase activity of v45a/q182l mutant on a/g was nearly undetectable at  <dig> nm .
with a/go substrate, both v45a and q182l mutants showed decrease in activity at  <dig>  and  <dig>  nm, respectively , whereas the v45a/q182l double mutant showed no activity at  <dig>  nm and decreased activity at  <dig>  and  <dig> nm .
the glycosylase activity assayed at the indicated active site concentrations is based on the active site titration results upon binding with a/go-dna.
since the mutants' glycosylase activities are affected differently from dna binding, thus, the active site concentrations measured by dna binding may be different from those measured by dna glycosylase activity.
we also used single-turnover glycosylase kinetics with enzyme in excess over dna  to compare the activities of wild-type and mutant ecmuty proteins.
as shown in figure  <dig>  the rates of cleavage on both a/g- and a/go-containing dna by muty-v45a were only slightly lower than those of the wild-type ecmuty protein.
however, the rates of cleavage of both a/g- and a/go-containing dna were much lower for q182l and v45a/q182l mutants than those of the wild-type ecmuty .
the rate constants  of q182l and v45a/q182l mutants with a/g mismatch at 37°c were  <dig>  ±  <dig>  min- <dig> and  <dig>  ±  <dig>  min- <dig>  respectively, and were about 10-fold lower that that of the wild-type ecmuty  .
the k <dig> of q182l and v45a/q182l mutants with a/go mismatch at 4°c were  <dig>  ±  <dig>  min- <dig> and  <dig>  ±  <dig>  min- <dig>  respectively, and were about 10-fold lower that that of the wild-type ecmuty  .
these results indicate that the overall repairing ability is reduced by q182l and v45a/q182l mutants but not by the v45a mutant.
the above results demonstrate that v45a and q182l mutations have significant effect on binding and glycosylase activities to a/g and a/go mispairs.
a catalytic domain of ecmuty with a v45n mutation has been shown to be inactive  <cit> .
these results indicate that v <dig> and q <dig> are significant for the adenine-specific activity of ecmuty.
ak <dig> values  are means ± standard deviation for more than three experiments.
k <dig> values with a/g and a/go are determined at 37°c and 20°c, respectively.
b the numbers in parenthesis are the folds of rate relative to wild-type  muty.
t/g and t/go binding and glycosylase activity
we next measured the t/g and t/go binding affinities of these mutants.
wild-type ecmuty showed a comparatively high kd  with a t/g mismatch , however, this value is much lower than that of ecmuty with c:g homoduplex   <cit> .
the kd values of v45a and q182l mutants to t/g drastically decreased to  <dig>  ±  <dig>  nm and  <dig>  ±  <dig>  nm, representing a  <dig> and 326-fold increase in binding affinity, respectively .
surprisingly, v45a/q182l double mutant showed a kd value of  <dig>  ±  <dig>  nm, which is close to that of wild-type ecmuty.
the geometry or architecture of the substrate binding pocket in the v45a/q182l double mutant may not favor thymine binding.
for t/go binding, v45a, q182l, and v45a/q182l mutants showed similar kd values as wild-type ecmuty , indicating that the t/go binding affinity of ecmuty does not involve v <dig> and q <dig>  this result is consistent with the findings of li et al.
<cit>  that tight t/go binding is controlled by the c-terminal domain of ecmuty.
because mig.mthi t/g glycosylase can be converted to an a/g glycosylase by replacing two residues   <cit> , we examined the glycosylase activities of vl45a, q182l, and v45a/q182l on t/g and t/go.
none of the ecmuty mutants showed any t/g and t/go glycosylase activity at enzyme concentration up to  <dig> μm under different buffers and temperatures .
therefore, recognition and glycosylase of ecmuty mutants to t/g and t/go are controlled by different mechanisms.
the previous success to mutate mig.mthi into muty-like enzyme  <cit>  is in contrast to our failure to covert ecmuty to thymine glycosylase.
it is possible that it is easier to catalyze adenine removal than thymine excision.
it is interesting to point out that wild-type mig.mthi is a weak adenine glycosylase and mig.mthi with a50v/l187q mutation becomes a stronger adenine glycosylase.
however, wild-type ecmuty has no thymine glycosylase activity and v45a/q182l mutant may be harder to gain this activity.
in addition, the active site of mig.mthi is more relaxed for both purine and pyrimidine excision while muty is more strict to excise purines.
the difference in the base removal by these two enzymes may be contributed by other residues surrounding the active site.
by aligning muty and mig.mthi sequences  and inspecting the structures of mig.mthi  <cit> , ecmuty catalytic domain  <cit>  with docked thymine , we predicted that several residues may contribute to their difference in activities.
the thymine docked into the ecmuty active site preserves the proposed edge-on hydrogen bonding interaction with the conserved glutamate  and can support a potential hydrogen bond with a tyrosine in a potential s120y mutant .
it is clear from figs.
2c and 2d that the muty active site poorly accommodates the methyl group of thymine relative to the mig.mthi active site.
in the present study, one important mutation  has been created.
based on our observations and the models shown in figure  <dig>  the subsequent mutations most likely to promote the conversion of muty into a thymine glycosylase would be q25r and l22f.
these two mutations would complete the methyl-binding pocket and stabilize the interaction with thymine.
in addition, an s120y mutation, which would provide an additional hydrogen bond  might further enhance any thymine glycosylase activity resulting from the q182l, q25r, and l22f mutations.
however, such predictions highlight the difficulty inherent in designing proteins with novel activities.
in particular, it has been shown that y126s mutation of mig.mthi inactivates t/g mismatch-specific glycosylase activity  <cit> , however, the presence of tyr in bsmuty indicates that tyr may not be the only key residue for t/g catalytic activity.
similarly, bsmuty contains r <dig> to accommodate the methyl group of thymine relative to the mig.mthi active site, but does not excise thymine.
recently, a bifuncional muty-like glycosylase, tthmuty, has been shown to have strong a/go and g/go activities, comparatively weak t/go and a/g activities, and no t/g glycosylase activity  <cit> .
unlike mig.mthi, tthmuty and ecmuty contain extra c-terminal domains which facilitate their recognition of the mispaired go base.
v <dig> and q <dig> of tthmuty are identical with the corresponding residues in ecmuty  but different from a <dig> and l <dig> of mig.mthi .
although tthmuty has weak t/go glycosylase activity, its l <dig> and q <dig> residues, which correspond to f <dig> and r <dig> of mig.mthi, respectively, are also conserved in ecmuty and bsmuty.
this suggests that f <dig> and r <dig> of mig.mthi may not be absolutely required for t/g or t/go glycosylase activity.
interestingly, y112s tthmuty mutant lost the thymine glycosylase activity supporting that y <dig> of mig.mthi may be important for t/g catalytic activity.
it remains to be tested whether s120y, q25r, and l22f mutants of ecmuty or in combination with v45a and q182l can gain t/g glycosylase activity.
v45a, q182l, and v45a/q182l ecmuty mutants cannot complement the muty mutation
the reduced binding and glycosylase activities toward a/g and a/go of these ecmuty mutants led us to check whether they have normal biological activity.
we examined the mutation frequency in rifampicin forward-mutation and lac+ reversion assays.
rifampicin binds to rna polymerase  and inhibits its transcriptional function.
mutations in the rifampicin binding site of rna polymerase results in cells' resistance to rifampicin.
in the absence of both ecmutm and ecmuty, a high level of rifampicin-resistant cells was observed .
the mutmmuty double mutant offers a more sensitive complementation assay than the muty single mutant.
expression of wild-type ecmuty complemented the mutmmuty double mutation .
expression of ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants partially complemented the deficient ecmutm and ecmuty function.
the mutation frequencies are  <dig>   <dig>  and  <dig> folds higher, respectively, compared to the cc <dig> wild-type .
a the strain cc <dig> mutm muty contains pet11a which is the expression vector for wild-type and mutant muty expression.
b-e the strain cc <dig> mutm muty expresses indicated muty protein cloned in the pet11a vector.
cc <dig> strain was designed to screen g:c→t:a transversions at an essential residue in the active site of β-galactosidase encoded by lacz gene, thus it allowed us to measure this type of mutation in ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants.
similar to the results of rifampicin forward assay, the mutation frequency of the mutmmuty double mutant increased 400-fold over the wild-type cells .
the ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants showed partial defect in preventing g:c→t:a transversions as compared to the wild-type ecmuty.
strains expressing v45a, q182l and v45a/q182l mutants, respectively, showed  <dig>   <dig>  and 17- fold higher mutation frequencies than cc <dig> wild-type .
these increased g:c→t:a mutation frequencies are in agreement with the reduced glycosylase activities with a/g and a/go mismatches in these mutants.
wild-type cells expressing the v45a mutant protein have an increased g:c to a:t mutation frequency
for the strains expressing v45a muty protein, the g:c→t:a mutation frequency measured by lac+  reversion assay was much lower than that measured by the rifampicin forward-mutation assay  .
because v45a mutant exhibits milder defect in adenine glycosylase activity than the other two mutants, we suspected that the v45a mutant with a 1273-fold increased t/g affinity  might prevent t/g mismatches from being repaired by other dna repair pathways.
to test this idea, we isolated genomic dna from rifampicin resistant colonies from cc <dig>  cells expressing ecmuty-v45a and identified base-base mutations of rpob gene  <cit> .
a significant increase in g:c→a:t transition was found in cells expressing ecmuty-v45a as compared to cells with vector alone .
for unknown reason, the mutation frequency of a:t to t:a was quite high in cc <dig> harboring vector and ecmuty-v45a.
cc <dig> strain was designed to screen g:c→a:t transitions in lacz gene.
thus, we further measured the g:c→a:t transition frequency by lac+ reversion assay in cc <dig>  strain expressing ecmuty-v45a.
the ecmuty-v45a expressing strain showed a  <dig> -fold higher lac+ reversion frequency.
a t-test of two sets of data of rows  <dig> and  <dig> in table  <dig> shows that this increase is statistically significant with p =  <dig>  .
these data are consistent with our postulation that ecmuty-v45a protein acts as a dominant-inhibitor of other dna repair pathways.
athe number in parenthesis are the percentages of each mutation type.
CONCLUSIONS
the substrate specificity of dna glycosylases is very subtle to the change of amino acids located in the dna binding pocket, and a single amino acid alteration may significantly alter its enzyme activity.
our results show that v <dig> and q <dig> of ecmuty are important for the binding affinity and glycosylase activity to dna containing a/g or a/go mismatches.
the v45a/q182l double mutant exhibited an additive reduction in binding to both the a/g and a/go as for the single mutants.
our unexpected results that ecmuty-v45a and ecmuty-q182l show increased t/g binding affinity without gaining t/g glycosylase indicate the complicated nature of the dna-enzyme interaction.

 reactive oxygen species are generated by endogenous processes such as mitochondrial oxidative phosphorylation as well as exogenously following exposure to ionizing radiation and chemicals. oxidative dna damage including strand breaks and oxidative base lesions are specifically repaired by base excision repair pathways. the first step in this pathway is carried out by a lesion-specific dna glycosylase, which cleaves the n-glycosidic bond between the base and deoxyribose sugar. the most abundant and highly mutagenic oxidative dna damage lesion is 8-oxo- -dihydroguanine that can form base-pair with adenine or cytosine during dna replication to produce a g:c to t:a transversion. in escherichia coli, mutt, mutm, muty, muts, and nei are involved in defending against the mutagenic effects of 8-oxog lesions [reviewed in and. the mutt protein has pyrophosphohydrolase activity, which eliminates 8-oxo-dgtp from the nucleotide pool. mutm glycosylase removes both mutagenic go adducts and ring-opened purine lesions paired with cytosines. muts and muty increase replication fidelity by removing the adenines misincorporated opposite go or g, and thus reduce g:c to t:a transversions. nei can excise go, when go is paired opposite a cytosine or adenine and can serve as a backup pathway to repair 8-oxog in the absence of mutm and muty. unlike most dna glycosylases, muty does not target a damaged base, but a normal base, e.g., removing a mispaired with go, c, and 5-hydroxyuracil. in addition, muty can excise guanines from g/go mismatches. muty also binds tightly to dna containing t/go and c/go, however, it exhibits no catalytic activity on them. the n-terminal domain of ecmuty retains the catalytic activity while the c-terminal domain of muty plays an important role in the recognition of go lesions. the x-ray crystal structures of ecmuty catalytic domain with bound adenine and intact bacillus stearothermophilus muty bound to dna show that muty distorts the bound dna substrate and the mismatched a is flipped out of the helix. in the active site pocket of ecmuty, seven amino acids are involved in adenine binding. v and q of ecmuty are proposed to be key residues in determining substrate specificity. v is considered to be important for glycosylase activity because v45n is inactive and is conserved among muty family members. q forms a hydrogen bond with adenine at the domain interface of ecmuty, but is less conserved. the n-terminal domain of muty shares similar structure, including the helix-hairpin-helix and gly/pro asp loop motifs, with alka, endoiii, and ogg the g/t mismatch glycosylase mig.mthi of methanobacterium thermoautotrophicim is a member of the hhh superfamily and is involved in reducing spontaneous deaminated bases from 5-methyl-cytosine residues. the substrate specificity of mig.mthi is in the order: u/g = t/g > g/g > t/c = u/c > a/g. in the binding pocket for the target base, mig.mthi shares six out of seven residues of ecmuty's adenine binding. the only non-conserved residue in this binding pocket is q of ecmuty, which corresponds to l of mig.mthi. interestingly, fondufe-mittendorf et al. could convert mig.mthi into a muty-like glycosylase with altered substrate preference of mig.mthi from t/g to a/g by replacing two residues in the substrate binding pocket. their data point to the potential importance of v and q in ecmuty substrate recognition. in this study, we have investigated the role of v and q of ecmuty by targeted mutagenesis. v45a, q182l, and v45a/q182l mutant proteins have reduced binding affinity and glycosylase activity to dna containing a/g or a/go mismatches. these mutants are impaired to complement e. coli muty mutants in vivo. the affinities to t/g dramatically increased in v45a and q182l mutants, but not in the v45a/q182l double mutant. however, v45a, q182l, and v45a/q182l mutant proteins do not exhibit glycosylase activity towards t/g or t/go. therefore, v and q of ecmuty are important for the adenine-specific activity of ecmuty.  selection of ecmuty mutation the study by fondufe-mittendorf et al. indicates that the substrate preference of mig.mthi can be altered from t/g to a/g by replacing two residues in the substrate binding pocket. their results are in agreement that v and q of ecmuty are the key determinants of adenine specificity. according to the adenine soaked ecmuty structure, v is located in the minor-groove reading α2–α motif and q is located in the adenine recognition α motif. interestingly, the proximity of v and q to adenine in ecmuty structure closely correlates with the bsmuty structure with the dna product and cleaved adenine. however, the residues corresponding to val and gln in d144n bsmuty with a/go-containing dna do not directly make contacts with the adenine of the a/go mismatch. in the later structure, the val seems to be positioned below the adenine, but the glu of bsmuty is far away from adenine. the fact that there are differences in adenine recognition among various muty structures raises a question whether these residues really are involved directly in adenine recognition. to determine whether these two residues are important for adenine recognition and excision, we carried out targeted mutagenesis study to determine the effect of ecmuty v45a, q182l, and double mutant on repair of a/g- and a/go-containing dna. we also further determined whether their substrate specificity is altered in terms of binding or catalysis of t/g or t/go mismatches. a/g and a/go binding and glycosylase activity we compared the binding affinities of v45a, q182l, and v45a/q182l double mutants with wild-type ecmuty to dna substrates containing a/g and a/go mismatches. first, we determined the active site concentrations of these proteins by performing a/go binding with dna concentrations which are at least 17-fold higher than their estimated dissociation constants. figure shows the average results from two independent experiments with nm of a/go-dna. the increase in wild-type ecmuty binding reached a maximum at a concentration about nm of muty and nm dna was bound. this horizontal line intercepts with the slope line of initial rate at nm of muty. thus, the wt ecmuty preparation is 68% active. similar analyses showed that v45a, q182l, and v45a/q182l were 48%, 25%, and 40% active, respectively. dissociation constant value for each mutant was determined with nine enzyme concentrations by at least three independent experiments. because muty displays a high affinity for its products, the kd values measure its affinities to a mixture of the substrate and the product. compared to wild-type ecmuty of which the kd to a/g is ± nm, the v45a and q182l mutants had kd values of ± and ± nm, respectively. the kd value of v45a/q182l double mutant was higher than the single mutants. thus, the affinities to a/g of v45a, q182l, and v45a/q182l are weakened by 5-, 7-, and 12-fold, respectively. for a/go binding, v45a and q182l mutations resulted in a 94- and 11-fold increase in kd values, respectively. the v45a/q182l double mutation resulted in a 102-fold increase in kd value. thus, v45a is more defective in a/go binding than a/g binding as compared to wild-type muty. it is interesting to note that the double mutant exhibited an additive reduction in binding to both the a/g and a/go as for the single mutants. these results confirm that v and q play important but independent roles in adenine binding. a kd values are mean ± standard deviation for more than three experiments using nine protein concentrations. bthe numbers in parenthesis are the folds of binding affinity relative to wild-type muty. because v45a and q182l ecmuty mutants showed weakened a/g and a/go affinities, we examined their glycosylase activities on a/g and a/go. v45a mutant showed decrease in a/g glycosylase activity at and nm concentrations while q182l mutant showed no activity at nm and decrease in activity at, and nm, as compared to wild-type muty. the glycosylase activity of v45a/q182l mutant on a/g was nearly undetectable at nm. with a/go substrate, both v45a and q182l mutants showed decrease in activity at and nm, respectively, whereas the v45a/q182l double mutant showed no activity at nm and decreased activity at and nm. the glycosylase activity assayed at the indicated active site concentrations is based on the active site titration results upon binding with a/go-dna. since the mutants' glycosylase activities are affected differently from dna binding, thus, the active site concentrations measured by dna binding may be different from those measured by dna glycosylase activity. we also used single-turnover glycosylase kinetics with enzyme in excess over dna to compare the activities of wild-type and mutant ecmuty proteins. as shown in figure the rates of cleavage on both a/g- and a/go-containing dna by muty-v45a were only slightly lower than those of the wild-type ecmuty protein. however, the rates of cleavage of both a/g- and a/go-containing dna were much lower for q182l and v45a/q182l mutants than those of the wild-type ecmuty. the rate constants of q182l and v45a/q182l mutants with a/g mismatch at 37°c were ± min- and ± min- respectively, and were about 10-fold lower that that of the wild-type ecmuty. the k of q182l and v45a/q182l mutants with a/go mismatch at 4°c were ± min- and ± min- respectively, and were about 10-fold lower that that of the wild-type ecmuty. these results indicate that the overall repairing ability is reduced by q182l and v45a/q182l mutants but not by the v45a mutant. the above results demonstrate that v45a and q182l mutations have significant effect on binding and glycosylase activities to a/g and a/go mispairs. a catalytic domain of ecmuty with a v45n mutation has been shown to be inactive. these results indicate that v and q are significant for the adenine-specific activity of ecmuty. ak values are means ± standard deviation for more than three experiments. k values with a/g and a/go are determined at 37°c and 20°c, respectively. b the numbers in parenthesis are the folds of rate relative to wild-type muty. t/g and t/go binding and glycosylase activity we next measured the t/g and t/go binding affinities of these mutants. wild-type ecmuty showed a comparatively high kd with a t/g mismatch, however, this value is much lower than that of ecmuty with c:g homoduplex. the kd values of v45a and q182l mutants to t/g drastically decreased to ± nm and ± nm, representing a and 326-fold increase in binding affinity, respectively. surprisingly, v45a/q182l double mutant showed a kd value of ± nm, which is close to that of wild-type ecmuty. the geometry or architecture of the substrate binding pocket in the v45a/q182l double mutant may not favor thymine binding. for t/go binding, v45a, q182l, and v45a/q182l mutants showed similar kd values as wild-type ecmuty, indicating that the t/go binding affinity of ecmuty does not involve v and q this result is consistent with the findings of li et al. that tight t/go binding is controlled by the c-terminal domain of ecmuty. because mig.mthi t/g glycosylase can be converted to an a/g glycosylase by replacing two residues, we examined the glycosylase activities of vl45a, q182l, and v45a/q182l on t/g and t/go. none of the ecmuty mutants showed any t/g and t/go glycosylase activity at enzyme concentration up to μm under different buffers and temperatures. therefore, recognition and glycosylase of ecmuty mutants to t/g and t/go are controlled by different mechanisms. the previous success to mutate mig.mthi into muty-like enzyme is in contrast to our failure to covert ecmuty to thymine glycosylase. it is possible that it is easier to catalyze adenine removal than thymine excision. it is interesting to point out that wild-type mig.mthi is a weak adenine glycosylase and mig.mthi with a50v/l187q mutation becomes a stronger adenine glycosylase. however, wild-type ecmuty has no thymine glycosylase activity and v45a/q182l mutant may be harder to gain this activity. in addition, the active site of mig.mthi is more relaxed for both purine and pyrimidine excision while muty is more strict to excise purines. the difference in the base removal by these two enzymes may be contributed by other residues surrounding the active site. by aligning muty and mig.mthi sequences and inspecting the structures of mig.mthi, ecmuty catalytic domain with docked thymine, we predicted that several residues may contribute to their difference in activities. the thymine docked into the ecmuty active site preserves the proposed edge-on hydrogen bonding interaction with the conserved glutamate and can support a potential hydrogen bond with a tyrosine in a potential s120y mutant. it is clear from figs. 2c and 2d that the muty active site poorly accommodates the methyl group of thymine relative to the mig.mthi active site. in the present study, one important mutation has been created. based on our observations and the models shown in figure the subsequent mutations most likely to promote the conversion of muty into a thymine glycosylase would be q25r and l22f. these two mutations would complete the methyl-binding pocket and stabilize the interaction with thymine. in addition, an s120y mutation, which would provide an additional hydrogen bond might further enhance any thymine glycosylase activity resulting from the q182l, q25r, and l22f mutations. however, such predictions highlight the difficulty inherent in designing proteins with novel activities. in particular, it has been shown that y126s mutation of mig.mthi inactivates t/g mismatch-specific glycosylase activity, however, the presence of tyr in bsmuty indicates that tyr may not be the only key residue for t/g catalytic activity. similarly, bsmuty contains r to accommodate the methyl group of thymine relative to the mig.mthi active site, but does not excise thymine. recently, a bifuncional muty-like glycosylase, tthmuty, has been shown to have strong a/go and g/go activities, comparatively weak t/go and a/g activities, and no t/g glycosylase activity. unlike mig.mthi, tthmuty and ecmuty contain extra c-terminal domains which facilitate their recognition of the mispaired go base. v and q of tthmuty are identical with the corresponding residues in ecmuty but different from a and l of mig.mthi. although tthmuty has weak t/go glycosylase activity, its l and q residues, which correspond to f and r of mig.mthi, respectively, are also conserved in ecmuty and bsmuty. this suggests that f and r of mig.mthi may not be absolutely required for t/g or t/go glycosylase activity. interestingly, y112s tthmuty mutant lost the thymine glycosylase activity supporting that y of mig.mthi may be important for t/g catalytic activity. it remains to be tested whether s120y, q25r, and l22f mutants of ecmuty or in combination with v45a and q182l can gain t/g glycosylase activity. v45a, q182l, and v45a/q182l ecmuty mutants cannot complement the muty mutation the reduced binding and glycosylase activities toward a/g and a/go of these ecmuty mutants led us to check whether they have normal biological activity. we examined the mutation frequency in rifampicin forward-mutation and lac+ reversion assays. rifampicin binds to rna polymerase and inhibits its transcriptional function. mutations in the rifampicin binding site of rna polymerase results in cells' resistance to rifampicin. in the absence of both ecmutm and ecmuty, a high level of rifampicin-resistant cells was observed. the mutmmuty double mutant offers a more sensitive complementation assay than the muty single mutant. expression of wild-type ecmuty complemented the mutmmuty double mutation. expression of ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants partially complemented the deficient ecmutm and ecmuty function. the mutation frequencies are and folds higher, respectively, compared to the cc wild-type. a the strain cc mutm muty contains pet11a which is the expression vector for wild-type and mutant muty expression. b-e the strain cc mutm muty expresses indicated muty protein cloned in the pet11a vector. cc strain was designed to screen g:c→t:a transversions at an essential residue in the active site of β-galactosidase encoded by lacz gene, thus it allowed us to measure this type of mutation in ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants. similar to the results of rifampicin forward assay, the mutation frequency of the mutmmuty double mutant increased 400-fold over the wild-type cells. the ecmuty-v45a, ecmuty-q182l and ecmuty-v45a/q182l mutants showed partial defect in preventing g:c→t:a transversions as compared to the wild-type ecmuty. strains expressing v45a, q182l and v45a/q182l mutants, respectively, showed and 17- fold higher mutation frequencies than cc wild-type. these increased g:c→t:a mutation frequencies are in agreement with the reduced glycosylase activities with a/g and a/go mismatches in these mutants. wild-type cells expressing the v45a mutant protein have an increased g:c to a:t mutation frequency for the strains expressing v45a muty protein, the g:c→t:a mutation frequency measured by lac+ reversion assay was much lower than that measured by the rifampicin forward-mutation assay. because v45a mutant exhibits milder defect in adenine glycosylase activity than the other two mutants, we suspected that the v45a mutant with a 1273-fold increased t/g affinity might prevent t/g mismatches from being repaired by other dna repair pathways. to test this idea, we isolated genomic dna from rifampicin resistant colonies from cc cells expressing ecmuty-v45a and identified base-base mutations of rpob gene. a significant increase in g:c→a:t transition was found in cells expressing ecmuty-v45a as compared to cells with vector alone. for unknown reason, the mutation frequency of a:t to t:a was quite high in cc harboring vector and ecmuty-v45a. cc strain was designed to screen g:c→a:t transitions in lacz gene. thus, we further measured the g:c→a:t transition frequency by lac+ reversion assay in cc strain expressing ecmuty-v45a. the ecmuty-v45a expressing strain showed a -fold higher lac+ reversion frequency. a t-test of two sets of data of rows and in table shows that this increase is statistically significant with p =. these data are consistent with our postulation that ecmuty-v45a protein acts as a dominant-inhibitor of other dna repair pathways. athe number in parenthesis are the percentages of each mutation type.  the substrate specificity of dna glycosylases is very subtle to the change of amino acids located in the dna binding pocket, and a single amino acid alteration may significantly alter its enzyme activity. our results show that v and q of ecmuty are important for the binding affinity and glycosylase activity to dna containing a/g or a/go mismatches. the v45a/q182l double mutant exhibited an additive reduction in binding to both the a/g and a/go as for the single mutants. our unexpected results that ecmuty-v45a and ecmuty-q182l show increased t/g binding affinity without gaining t/g glycosylase indicate the complicated nature of the dna-enzyme interaction. 
v45a, q182l, and v45a/q182l ecmuty mutants could not reduce the g:c to t:a mutation frequency of a muty mutant. the substrate specificities are altered in v45a, q182l, and v45a/q182l ecmuty mutants. however, in contrast to a previous report that mig.mthi thymine dna glycosylase can be converted to a muty-like adenine glycosylase by replacing two residues, both v45a and q182l ecmuty mutants did not exhibit any t/g or t/8-oxog glycosylase activity. targeted mutagenesis study was performed to determine the substrate specificities of v45a, q182l, and v45a/q182l double mutant of ecmuty. the double mutant exhibited an additive reduction in binding to both the a/g and a/go in comparison to the single mutants. surprisingly, the v45a/q182l double mutant had similar binding affinities to t/g as the wild-type ecmuty. both residues are spatially close to each other in the active site and are conserved in muty family proteins but not in methanobacterium thermoautotrophicum mig.mthi t/g mismatch dna glycosylase. expression of the v45a mutant protein caused a dominant negative phenotype with an increased g:c to a:t mutation frequency. both v45a and q182l mutants had substantially increased affinities towards t/g, however, they did not exhibit any t/g or t/8-oxog glycosylase activity. v45a and q182l mutants had reduced binding and glycosylase activities for a/g and a/8-oxog mismatches and increased affinities towards t/g mismatch. the dominant negative phenotype of v45a ecmuty mutant protein is probably caused by its increased binding affinity to t/g mismatch and thus inhibiting other repair pathways. v and q of ecmuty are considered to be the key determinants of adenine specificity. all three mutants had significantly lower binding and glycosylase activities for a/g and a/8-oxog mismatches than the wild-type enzyme. these mutants were also tested for binding and glycosylase activities with t/g- or t/8-oxog-containing dna.
 escherichia coli muty reduces mutagenesis by removing adenines paired with guanines or -dihydro-8-oxo-guanines. v and q of ecmuty are considered to be the key determinants of adenine specificity. both residues are spatially close to each other in the active site and are conserved in muty family proteins but not in methanobacterium thermoautotrophicum mig.mthi t/g mismatch dna glycosylase.  targeted mutagenesis study was performed to determine the substrate specificities of v45a, q182l, and v45a/q182l double mutant of ecmuty. all three mutants had significantly lower binding and glycosylase activities for a/g and a/8-oxog mismatches than the wild-type enzyme. the double mutant exhibited an additive reduction in binding to both the a/g and a/go in comparison to the single mutants. these mutants were also tested for binding and glycosylase activities with t/g- or t/8-oxog-containing dna. both v45a and q182l mutants had substantially increased affinities towards t/g, however, they did not exhibit any t/g or t/8-oxog glycosylase activity. surprisingly, the v45a/q182l double mutant had similar binding affinities to t/g as the wild-type ecmuty. v45a, q182l, and v45a/q182l ecmuty mutants could not reduce the g:c to t:a mutation frequency of a muty mutant. expression of the v45a mutant protein caused a dominant negative phenotype with an increased g:c to a:t mutation frequency.  the substrate specificities are altered in v45a, q182l, and v45a/q182l ecmuty mutants. v45a and q182l mutants had reduced binding and glycosylase activities for a/g and a/8-oxog mismatches and increased affinities towards t/g mismatch. however, in contrast to a previous report that mig.mthi thymine dna glycosylase can be converted to a muty-like adenine glycosylase by replacing two residues, both v45a and q182l ecmuty mutants did not exhibit any t/g or t/8-oxog glycosylase activity. the dominant negative phenotype of v45a ecmuty mutant protein is probably caused by its increased binding affinity to t/g mismatch and thus inhibiting other repair pathways. 