Role of DNA base excision repair in the mutability and virulence of Streptococcus mutans

The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defences for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H(2) O(2) ), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the Galleria mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared with the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.     

Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

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.     

Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site

The formation of clustered DNA damage sites is a unique feature of ionizing radiation. Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites. Using a plasmid-based method, damaged DNA containing uracil positioned at 1–5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY. Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions. The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid. In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site. Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC→TA transversions. The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis.     

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.     

Removal of hydantoin products of 8-oxoguanine oxidation by the Escherichia coli DNA repair enzyme, FPG

An intriguing feature of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is that it is highly reactive toward further oxidation. Indeed, OG has been shown to be a "hot spot" for oxidative damage and susceptible to oxidation by a variety of cellular oxidants. Recent work has identified two new DNA lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), resulting from one-electron oxidation of OG. The presence of Gh and Sp lesions in DNA templates has been shown to result in misinsertion of G and A by DNA polymerases, and therefore, both are potentially mutagenic DNA lesions. The base excision repair (BER) glycosylases Fpg and MutY serve to prevent mutations associated with OG in Escherichia coli, and therefore, we have investigated the ability of these two enzymes to process DNA duplex substrates containing the further oxidized OG lesions, Gh and Sp. The Fpg protein, which removes OG and a variety of other oxidized purine base lesions, was found to remove Gh and Sp efficiently opposite all four of the natural DNA bases. The intrinsic rate of damaged base excision by Fpg was measured under single-turnover conditions and was found to be highly dependent upon the identity of the base opposite the OG, Gh, or Sp lesion; as expected, OG is removed more readily from an OG:C- than an OG:A-containing substrate. However, when adenine is paired with Gh or Sp, the rate of removal of these damaged lesions by Fpg was significantly increased relative to the rate of removal of OG from an OG:A mismatch. The adenine glycosylase MutY, which removes misincorporated A residues from OG:A mismatches, is unable to remove A paired with Gh or Sp. Thus, the activity of Fpg on Gh and Sp lesions may dramatically influence their mutagenic potential. This work suggests that, in addition to OG, oxidative products resulting from further oxidation of OG should be considered when evaluating oxidative DNA damage and its associated effects on DNA mutagenesis.     

MutY is down-regulated by oxidative stress in E. coli

In Escherichia coli, MutM (8-oxoG DNA glycosylase/lyase or Fpg protein), MutY (adenine DNA glycosylase) and MutT (8-oxodGTPase) function cooperatively to prevent mutation due to 7, 8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic oxidative DNA adduct. MutM activity has been demonstrated to be induced by oxidative stress. Its regulation is under the negative control of the global regulatory genes, fur, fnr and arcA. However, interestingly the presence of MutY increases the mutation frequency in mutT- background because of MutY removes adenine (A) from 8-oxoG:A which arises from the misincorporation of 8-oxoG against A during DNA replication. Accordingly we hypothesized that the response of MutY to oxidative stress is opposite to that of MutM and compared the regulation of MutY activity with MutM under various oxidative stimuli. Unlike MutM, MutY activity was reduced by oxidative stress. Its activity was reduced to 30% of that of the control when E. coli was treated with paraquat (0.5 mM) or H

2

O

2

(0.1 mM) and induced under anaerobic conditions to more than twice that observed under aerobic conditions. The reduced mRNA level of MutY coincided with its reduced activity by paraquat treatment. Also, the increased activity of MutY in anaerobic conditions was reduced further in E. coli strains with mutations in fur, fnr and arcA and the maximum reduction in activity was when all mutations were present in combination, indicating that MutY is under the positive control of these regulatory genes. Therefore, the down-regulation of MutY suggests that there has been complementary mechanism for its mutagenic activity under special conditions. Moreover, the efficacy of anti-mutagenic action should be enhanced by the reciprocal co-regulation of MutM.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

MutY is Down-regulated by Oxidative Stress in E. coli

In Escherichia coli, MutM (8-oxoG DNA glycosylase/lyase or Fpg protein), MutY (adenine DNA glycosylase) and MutT (8-oxodGTPase) function cooperatively to prevent mutation due to 7, 8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic oxidative DNA adduct. MutM activity has been demonstrated to be induced by oxidative stress. Its regulation is under the negative control of the global regulatory genes, fur, fnr and arcA. However, interestingly the presence of MutY increases the mutation frequency in mutT- background because of MutY removes adenine (A) from 8-oxoG:A which arises from the misincorporation of 8-oxoG against A during DNA replication. Accordingly we hypothesized that the response of MutY to oxidative stress is opposite to that of MutM and compared the regulation of MutY activity with MutM under various oxidative stimuli. Unlike MutM, MutY activity was reduced by oxidative stress. Its activity was reduced to 30% of that of the control when E. coli was treated with paraquat (0.5 mM) or H₂O₂ (0.1 mM) and induced under anaerobic conditions to more than twice that observed under aerobic conditions. The reduced mRNA level of MutY coincided with its reduced activity by paraquat treatment. Also, the increased activity of MutY in anaerobic conditions was reduced further in E. coli strains with mutations in fur, fnr and arcA and the maximum reduction in activity was when all mutations were present in combination, indicating that MutY is under the positive control of these regulatory genes. Therefore, the down-regulation of MutY suggests that there has been complementary mechanism for its mutagenic activity under special conditions. Moreover, the efficacy of anti-mutagenic action should be enhanced by the reciprocal co-regulation of MutM.     

Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase

The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.     

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k_obs of over 2500 min^?1.     

A Novel Role for Escherichia coli Endonuclease VIII in Prevention of Spontaneous G→T Transversions

In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.     

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.     

Interaction of DNA containing Fapy.dA or its C-nucleoside analogues with base excision repair enzymes. Implications for mutagenesis and enzyme inhibition

Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.     

Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair

Background  

Neisseria meningitidis, the causative agent of meningococcal disease, is exposed to high levels of reactive oxygen species inside its exclusive human host. The DNA glycosylase Fpg of the base excision repair pathway (BER) is a central player in the correction of oxidative DNA damage. This study aimed at characterizing the meningococcal Fpg and its role in DNA repair.     

Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 A resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 A) and Nei-R252A (2.05 A). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a approximately 50 degrees rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.     

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O₂. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.     

Escherichia coli MutY and Fpg utilize a processive mechanism for target location

MutY and formamidopyrimidine-DNA-glycosylase (Fpg) are base-excision repair (BER) enzymes involved in the 8-oxoguanine repair pathway in Escherichia coli. An impressive feature of these enzymes is the ability to locate 8-oxoguanine lesions among a large excess of undamaged DNA. To provide insight into the mechanism of target location, the ability of these enzyme to utilize a one-dimensional processive search (DNA sliding) or distributive (random diffusion-mediated) mechanism was investigated. Each enzyme was incubated with double-stranded concatemeric polynucleotides containing a site-specific target site at 25-nucleotide (nt) intervals. The products of each reaction were analyzed after further treatment and denaturation. A rapid accumulation of predominantly 25-nt fragments would indicate the utilization of a processive mechanism, whereas oligomeric multiples of 25-nt fragments would form if a distributive mechanism were used. Both Fpg and MutY were found to function processively on concatemers containing 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG).C and G.A mispairs, respectively. An increase in sodium chloride concentration results in the modulation from a processive to distributive mechanism for both enzymes. Interestingly, processive behavior was not observed in the reaction of MutY with concatemers containing OG.A mispairs. A truncated form of MutY (Stop 225) containing only the N-terminal domain was found to behave in a manner consistent with a processive mechanism with both OG.A- and G.A-containing substrates. This suggests that the C-terminal domain of MutY plays an important role in the mechanism by which the enzyme detects OG.A base pairs in DNA.     

A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity

The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.