Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.     

Helicobacter pylori genes involved in avoidance of mutations induced by 8-oxoguanine

Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.     

Molecular cloning and functional analysis of the MutY homolog of Deinococcus radiodurans

The mutY homolog gene (mutY(Dr)) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutY(Ec)) protein. Expressed MutY(Dr) is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY(Dr) with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY(Ec) protein, purified recombinant MutY(Dr) expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY(Dr) exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY(Dr) at the position corresponding to the position of MutY(Ec) Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY(Dr) are similar to those of MutY(Ec). Although MutY(Dr) has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY(Ec) does, the binding affinities for both mismatches are slightly lower for MutY(Dr) than for MutY(Ec). Thus, MutY(Dr) can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.     

Physical and functional interactions between Escherichia coli MutY glycosylase and mismatch repair protein MutS

Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSalpha (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.     

The C-terminal domain of MutY glycosylase determines the 7,8-dihydro-8-oxo-guanine specificity and is crucial for mutation avoidance

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, or A/C mismatches and also has a weak guanine glycosylase activity on G/8-oxoG-containing DNA. The N-terminal domain of MutY, residues 1-226, has been shown to retain catalytic activity. Substrate binding, glycosylase, and Schiff base intermediate formation activities of the truncated and intact MutY were compared. MutY has high binding affinity with 8-oxoG when mispaired with A, G, T, C, or inosine. The truncated protein has more than 18-fold lower affinities for binding various 8-oxoG-containing mismatches when compared with intact MutY. MutY catalytic activity toward A/8-oxoG-containing DNA is much faster than that on A/G-containing DNA whereas deletion of the C-terminal domain reduces its catalytic preference for A/8-oxoG-DNA over A/G-DNA. MutY exerts more inhibition on the catalytic activity of MutM (Fpg) protein than does truncated MutY. The tight binding of MutY with GO mispaired with T, G, and apurinic/apyrimidinic sites may be involved in the regulation of MutM activity. An E. coli mutY strain that produces an N-terminal 249-residue truncated MutY confers a mutator phenotype. These findings strongly suggest that the C-terminal domain of MutY determines the 8-oxoG specificity and is crucial for mutation avoidance by oxidative damage.     

Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.     

Hyperrecombination in Streptococcus pneumoniae depends on an atypical mutY homologue

The unusual behavior of the mutation ami36, which generates hyperrecombination in two point crosses, was previously attributed to a localized conversion process changing A/G mispairs into CG pairs. Although the mechanism was found to be dependent on the DNA polymerase I, the specific function responsible for this correction was still unknown. Analysis of the pneumococcal genome sequence has revealed the presence of an open reading frame homologous to the gene mutY of Escherichia coli. The gene mutY encodes an adenine glycosylase active on A/G and A/7,8-dihydro-8-oxoguanine (8-OxoG) mismatches, inducing their repair to CG and C/8-OxoG, respectively. Here we report that disrupting the pneumococcal mutY homologue abolishes the hyperrecombination induced by ami36 and leads to a mutator phenotype specifically enhancing AT-to-CG transversions. The deduced amino acid sequence of the pneumococcal MutY protein reveals the absence of four cysteines, highly conserved in the endonuclease III/MutY glycosylase family, which ligate a [4Fe-4S](2+) cluster. The actual function of this cluster is still intriguing, inasmuch as we show that the pneumococcal gene complements a mutY strain of E. coli.     

Fission yeast (Schizosaccharomyces pombe) cells defective in the MutY-homologous glycosylase activity have a mutator phenotype and are sensitive to hydrogen peroxide

The modified base 7,8-dihydro-8-oxo-guanine (8-oxoG) is one of the most stable deleterious products of oxidative DNA damage because it mispairs with adenine during DNA replication. In the fission yeast Schizosaccharomyces pombe, the MutY homolog (SpMYH) is responsible for removing misincorporated adenines from A/8-oxoG or A/G mismatches and thus preventing G:C to T:A mutations. In order to study the functional role of SpMYH, an SpMYH knockout strain was constructed. The SpMYH knockout strain, which does not express SpMYH and has no A/8-oxoG glycosylase activity, displays a 36-fold higher frequency of spontaneous mutations than the wild type strain. Disruption of SpMYH causes increased sensitivity to H2O2 but not to UV-irradiation. Expression of SpMYH in the mutant cells restores the adenine glycosylase activity, reduces the mutation frequency, and elevates the resistance to H2O2. Asp172 of SpMYH is conserved in a helix-hairpin-helix superfamily of glycosylases. The SpMYHA strain expressing D172N SpMYH retained the mutator phenotype. Moreover, when D172N mutant SpMYH was expressed in the wild-type cells, the mutation frequency observed was even higher than that of the parental strains. Thus, a mutant SpMYH that retains substrate-binding activity but is defective in glycosylase activity exhibits a dominant negative effect. This is the first demonstration that a MutY homolog plays an important role in protecting cells against oxidative DNA damage in eukaryotes.     

Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E.coli enzymes

The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes. In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.     

Regulation of oxidative DNA damage repair: The adenine:8-oxo-guanine problem

Reactive oxygen species (ROS) constantly attack DNA. One of the best-characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). Many human diseases, such as cancer and neurodegenerative disorders, have been correlated with oxidative DNA damage. In the last few years, DNA polymerase (Pol) λ, one of the 15 cellular Pols, has been identified to play an important role in performing accurate translesion synthesis over 8-oxo-G. This is eminently important, since normally faithful replicative Pols α, δ and ε, with their tight active center, often wrongly incorporate adenine (A) opposite the 8-oxo-G lesion. A:8- oxo-G mispairs are accurately repaired by the pathway identified in our laboratory involving MutY DNA glycosylase homolog (MutYH) and Pol λ. Until now, very little was known about the spatial and temporal regulation of Pol λ and MutYH in active repair complexes. We now showed in our latest publication that the E3 ligase Mule can ubiquitinate and degrade Pol λ, and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. In contrast, phosphorylation of Pol λ by Cdk2/cyclinA counteracts this degradation by recruiting it to MutYH on chromatin to form active 8-oxo-G repair complexes.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

Escherichia coli MutY has an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA by excising adenines from OG.A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG.A substrate on the kinetics of base removal, mismatch affinity and repair to G-C in an E. coli-based assay. Notably, adenine modification was tolerated in the cellular assay, whereas modification of OG resulted in minimal cellular repair. High affinity for the mismatch and efficient base removal required the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature that is necessary for MutY to locate OG.A mismatches and select the appropriate adenines for excision to initiate repair in vivo before replication.     

Regulation of Human MutYH DNA Glycosylase by the E3 Ubiquitin Ligase Mule

Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.     

Characterization of an Escherichia coli mutant MutY with a cysteine to alanine mutation at the iron-sulfur cluster domain

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxoguanine (8-oxoG). The [4Fe-4S] cluster of MutY is ligated by four conserved cysteine residues and has been shown to be important in substrate recognition. Here, we show that the C199A mutant MutY is very insoluble and can be denatured and renatured to regain activity only if iron and sulfur are present in the renaturation steps. The solubility of C199A-MutY can be improved substantially as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. Here, we describe the first biochemical characterization of the purified GB1-C199A-MutY protein which contains a [3Fe-4S] cluster. The apparent dissociation constant (K(d)) values of GB1-C199A-MutY with both A/G and A/8-oxoG mismatches are slightly higher than that of the wild-type protein. The DNA glycosylase activity of GB1-C199A-MutY is comparable to that of the wild-type enzyme. Interestingly, the major difference between the C199A-MutY and wild-type proteins is their trapping activities (formation of Schiff base intermediates). The GB1-C199A-MutY mutant has a weaker trapping activity than the wild-type enzyme. Importantly, highly expressed GB1-C199A-MutY and untagged C199A-MutY can complement mutY mutants; however, GB1-C199A-MutY and untagged C199A-MutY cannot complement mutY mutants in vivo when both proteins are poorly expressed. Therefore, an intact [4Fe-4S] cluster domain is critical for MutY stability and activity.     

Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

The active site of the Escherichia coli MutY DNA adenine glycosylase.

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial. To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches. Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates. However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA. Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation.     

Mutagenic effects of gamma-rays and incorporated 8-3H-purines on extracellular lambda phage: influence of mutY and mutM host mutations

The lethal and mutagenic effects on phage lambdacI857 of 60Co gamma-rays and of decay of 3H incorporated into phage DNA both as 8-3H-deoxyadenosine and 8-3H-deoxyguanosine (using 8-3H-adenine as a labelled DNA precursor) were studied on four isogenic Escherichia coli strains: AB1157 M(+)Y(+) (wild type, mutM(+) mutY(+)), AB1157 M(-)Y(+) (mutM::kan mutY(+) mutant deficient in the formamidopyrimidine-DNA glycosylase MutM), AB1157 M(+)Y(-) (mutM(+) mutY mutant deficient in the A:G mismatch DNA glycosylase MutY), and AB1157 M(-)Y(-) (mutM::kan mutY double mutant deficient in both DNA glycosylases). The main products of transmutation component of 3H decay in position 8 of purine residues are 8-oxo-7, 8-dihydroadenine (8-oxoA) and 8-oxo-7,8-dihydroguanine (8-oxoG), the latter being responsible for the most part of the mutagenic effect. The lethal effects of both gamma-rays and tritium decay virtually did not depend on the repair phenotypes of the host strains used. Therefore, the MutM and MutY glycosylases are not involved in the repair of lethal DNA damages induced by ionizing radiation or by the transmutation component of 3H decay in purine residues of phage DNA. The efficiencies of mutagenic action of 3H-purines E(m) (frequencies of c-mutations per one 3H decay in phage genome) were 2.4-, 3.8- and 55-fold higher in the M(-)Y(+), M(+)Y(-) and M(-)Y(-) mutants, respectively, in comparison to the wild-type host. The mutagenic efficiencies E(m) for gamma-rays were nearly identical in the M(+)Y(+) and M(-)Y(+) hosts, but were increased 1.8- and 8.3-fold, respectively, in the M(+)Y(-) and M(-)Y(-) mutants. These data suggest that: (1) the MutY and MutM DNA glycosylases are important for prevention of mutations caused not only by spontaneous oxidation of guanine residues, but also by ionizing radiation or by decay of 3H incorporated into purine bases of DNA; (2) the MutY and MutM enzymes functionally cooperate in elimination of mutagenic damages induced by these agents.     

An Escherichia coli MutY mutant without the six-helix barrel domain is a dimer in solution and assembles cooperatively into multisubunit complexes with DNA

Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO). MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpin-helix motif, and a C-terminal domain. Here, we demonstrate that the mutant MutY(Delta26-134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo. However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations. The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20- and 44-bp DNA fragments, respectively, in a concentration-dependent manner. Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA. Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative. This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains. The nonspecific binding of MutY(Delta26-134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch.