The currently known substrates for MutY and MUTYH are summarized in Table 1. Analysis of the substrate specificity for MutY demonstrated that it acts as a glycosylase on A:G, A:8-oxo-G, A:C, and A:8-oxo-A mismatches, always removing the undamaged A from each substrate (Michaels et al., 1992b). Lu et al. further refined the DNA determinants and substrate specificities for the catalytic activity of MutY, using binding and endonuclease assays with a variety of different A-containing mismatches, and concluded that DNA sequences proximal to the mismatch as well as specific functional groups of mismatched bases dictate the recognition and catalysis by MutY (Lu et al., 1995). Moreover, while MutY bound the A:8-oxo-G much tighter than A:G, its activity on A:8-oxo-G was weaker than on A:G mismatches. Bulychev et al. contradicted this notion in a subsequent report stating that A:8-oxo-G appeared to be the natural substrate for MutY, as judged by the specificity constants and the fact that the presence of an 8-oxo-group in G increased significantly the rate of removal of A from all tested substrates (Bulychev et al., 1996). Additionally to A:8-oxo-G, MutY was shown to bind to G:8-oxo-G mismatches as well, and it was capable of removing G from this substrate (Zhang et al., 1998). The sequence context surrounding an A:G mismatch was shown to also significantly influence the catalytic activity of MutY (Sanchez et al., 2003).

8-oxo-G is chemically labile toward further oxidation into guanidinohydantoin (Sp1), spiroiminodihydantoin (Sp2), oxaluric acid, and urea. Delaney et al. investigated the activity of MutY on these lesions by introducing them into single-stranded viral genomes which were replicated in E. coli proficient or deficient for MutY (Delaney et al., 2007). These lesions were found to be equally mutagenic in terms of frequency in both genetic backgrounds and to yield similar mutation spectra, suggesting that MutY does not play a role in the excision of these bases. Interestingly Sp1 and Sp2 were more toxic to the cells that were proficient in MutY.

2-hydroxyadenine (2-OH-A) is a lesion that is induced by Fenton-type ROS and is produced for instance by H₂O₂ treatment of cultured mammalian cells (Jaruga and Dizdaroglu, 1996). Incorporation of 2-OH-dATP into the bacterial genome by pol III was shown to yield slightly increased mutant frequencies in a MutY deficient background in E. coli, suggesting that the processing of 2-OH-A damage possibly also involves the action of MutY (Kamiya and Kasai, 2000a). However, follow-up work by the same authors showed that, irrespectively of the base in the complementary strand, DNA with 2-OH-A presented a very poor substrate for MutY, and therefore illustrated that neither MutY nor Fpg seemed to play a role in 2-OH-A removal from DNA (Kamiya and Kasai, 2000b). Another result by Hashiguchi et al. again reassessed this finding and they reported that MutY indeed bound to 2-OH-A in duplex with G, A, or C and displayed a DNA glycosylase activity capable of removing 2-OH-A from 2-OH-A:G mismatches, which was dependent on the C-terminal domain of the protein (Hashiguchi et al., 2002).

FapyG is a DNA lesion that arises from oxidative stress by ring-fragmentation of the purine base. MutY excised A from A:FapyG mismatches, and this reaction was faster than the removal of A from A:G, but still slower than that from A:8-oxo-G in vitro (Wiederholt et al., 2003).

One group reported that MutY efficiently recognized 7-deaza-2′-deoxyadenosine (Z) and its non-polar isostere 4-methylindole-beta-deoxynucleoside (M) opposite 8-oxo-G and G in DNA, with a preference for M:8-oxo-G over Z:8-oxo-G mispairs (Chepanoske et al., 2000b). This finding was contradicting a previous report, in which Z:G mispairs were neither bound nor processed by MutY (Lu et al., 1995).

Lu et al. showed that MutY competes with and inhibits endonuclease VIII on its natural substrate, the hydroxyurea (hoU):A mismatch (Lu et al., 2006b).

A MutY variant from Thermus thermophilus processed A:8-oxo-G, G:8-oxo-G as well as T:8-oxo-G and A:G mismatches, but in contrast to other MutY variants, was shown to harbor a bifunctional glycosylase activity (Back et al., 2006).

The cloning of E. coli MutY revealed that it shared significant sequence homology to the bacterial endonuclease III (EndoIII), which acts on damaged base pairs (Michaels et al., 1990). MutY was shown to be an iron-sulfur (Fe-S) cluster protein containing both N-glycosylase and a 3′ AP endonuclease activity (Tsai-Wu et al., 1992). Initially there was some confusion regarding the enzymatic activity of MutY. While some reports stated that MutY also acted as an endonuclease on AP sites, therefore functioning as a bifunctional glycosylase (Tsai-Wu et al., 1992; Lu et al., 1995, 1996; Gogos et al., 1996; Manuel and Lloyd, 1997), Zharkov and Grollman showed that MutY does not harbor any AP lyase activity (Zharkov and Grollman, 1998). They hypothesized that the previous observations for the observed AP-activity were rather caused by heat-induced cleavage of the AP site and not due to an actual enzymatic activity. Moreover, this report suggested that the tight binding of MutY to its DNA substrate prevented the access of another bacterial glycosylase, the formamidopyrimidine-DNA glycosylase (Fpg), to the substrate. Consequently, MutY seemed to prevent a possible generation of a DNA double-strand break (DSB) by Fpg and thus possibly to play a role in the regulation of BER.

When considering the catalytic activity of MutY (or any other DNA glycosylase), it is important to keep in mind that the catalytic cycle can be roughly subdivided into different stages, namely (1) recognition and binding of the enzyme to the substrate, (2) hydrolysis of the N-glycosidic bond or base-excision, and (3) dissociation of the enzyme or release of the resulting AP site. We have tried to structure the discussion according to these three steps in the catalytic cycle, whenever possible.

Substrate recognition. Multiple studies elucidating the contributions of the different parts of the MutY protein have been undertaken. Proteolytic digestion of MutY with thermolysin produced two fragments, an N-terminal one of 25 kDa and a C-terminal one of 12 kDa, respectively (Gogos et al., 1996). While the 12 kDa fragment did not display any detectable enzymatic activity, it was found to play an important role in the repair of mismatched oxidized DNA, as its deletion significantly impaired the binding and activity of MutY on A:8-oxo-G substrates, while it did not influence binding and cleavage of A:G substrates. On the other hand, a similar study, generating a 26 kDa N-terminal domain of MutY by trypsin-mediated proteolysis showed that this 26 kDa subunit was catalytically active, contained both DNA glycosylase and AP lyase activity, and was functionally identical with the full-length protein (Manuel et al., 1996; Manuel and Lloyd, 1997). A 14 kDa C-terminal domain of MutY (AA 1–226) was demonstrated to be the principal determinant for 8-oxo-G specificity, as its deletion remarkably enhanced the dissociation of the enzyme from A:8-oxo-G and reduced the rate of A removal from these substrates compared to A:G mismatches (Noll et al., 1999). This was interpreted such that the C-terminal domain facilitated A base flipping. Also, this study found that the C-terminal domain of MutY showed homology with MutT, suggesting that it might serve in 8-oxo-G recognition. Another report supported this view by showing that the N-terminal domain of MutY (AA 1–226) had a 18-fold lower affinity for binding various 8-oxo-G mismatches, a reduced catalytic preference for A:8-oxo-G over A:G mismatches and exhibited a lower inhibition on Fpg activity than the wild-type (wt) MutY (Li et al., 2000). These results suggested that the C-terminal domain of the protein determines its 8-oxo-G specificity and is crucial for mutation avoidance. The C-terminal domain was then shown to mediate additional contacts between MutY and A:8-oxo-G containing substrates that are not found in interaction with A:G (Li and Lu, 2000), thereby promoting the efficient recognition of substrates by MutY (Chmiel et al., 2001) and also affecting the catalytic activities toward A:G mismatches (Li and Lu, 2003). Taken together, the C-terminal domain of MutY seems to contribute substantially to the A:8-oxo-G substrate recognition.

It is still not entirely clear, how MutY is capable to efficiently recognize all its substrates from among the vast amount of undamaged base pairs. Along this line, the Fe-S cluster present in MutY was shown to be critical for the specific recognition of its DNA substrate and its enzymatic activity (Porello et al., 1998a; Golinelli et al., 1999; Chepanoske et al., 2000a). It has also been suggested that the relative oxidation resistance of the Fe-S cluster may be an important aspect to guarantee the activity of MutY under conditions of oxidative stress (Messick et al., 2002). K142 in MutY, earlier shown to be involved in formation of tight interactions with DNA, was shown to make specific contacts with 8-oxo-G, and DNA-mediated charge transport (CT) was suggested as signal to promote the binding of MutY to DNA from a distance (Boon et al., 2002). Along this line, DNA-mediated CT led to oxidation of DNA-bound MutY, suggesting that G radicals provide the signal to stimulate DNA repair by the redox activation of DNA repair proteins through CT (Yavin et al., 2005). Further substantiating this idea, Boal et al. proposed that the rapid redistribution of proteins to the sites of DNA damage was mediated through redox activation involving the Fe-S clusters in proteins such as MutY and EndoIII (Boal et al., 2005; Yavin et al., 2006). A theoretical study of the DNA damage recognition by Bacillus stearothermophilus MutY proposed that the CT from MutY to DNA through hole transfer, which is specially efficient near an 8-oxo-G, leads to the stabilization of the enzyme in a conformation required for recognition of the lesion (Lin et al., 2008). Examination of the charge-transfer model by atomic force microscopy further validated this concept and emphasized the possibility that indeed repair proteins might be recruited to DNA lesions by DNA-mediated CT in the cellular context (Boal et al., 2009). The authors therefore proposed a model wherein the binding of Fe-S cluster containing DNA repair proteins (such as MutY and EndoIII) to DNA activates them toward oxidation. First, the formation of a guanine radical oxidizes a repair protein bound to DNA and thus stabilizes the binding of this protein. This step is followed by the binding of a second protein near the first one. Because also this protein gets oxidized during binding and transfers an electron to the DNA, it will induce a DNA-mediated CT from the second to the first protein if no damage is present in the DNA stretch between the two binding sites. This CT leads to reduction of the first protein and thus to its release from DNA, because in the reduced state it has a lower affinity to DNA. However, if there is a DNA lesion between the two bound proteins, the CT does not take place (it is “blocked” by the intervening lesion). In this situation both of the proteins remain bound and can subsequently catalyze repair steps. Through examination of CT mutants of EndoIII the group subsequently linked the ability of a repair protein to carry out DNA CT and its ability to localize to damaged DNA and thus further underlined their model (Romano et al., 2011). Taken together, the role for the Fe-S cluster as redox cofactor to search for damaged bases using DNA-mediated CT becomes more and more substantiated and really presents a plausible scenario to explain the mechanisms of full-genome search for lesions.

Base-excision. Investigations into the glycosylase activity of MutY revealed a distinctive difference in the processing of A:8-oxo-G compared to A:G mismatches (Porello et al., 1998b). Hydrolysis of A from opposite 8-oxo-G was at least 6-fold faster than from the A:G mispair. Interestingly however, MutY “lingered” when excising from an A:8-oxo-G base pair and released the product with a much slower kinetic compared to the A:G substrate. This delay in substrate release might protect 8-oxo-G from being prematurely accessed and removed by other glycosylases, as also suggested by Zharkov and Grollman (1998). A detailed study of the active site revealed the importance of several amino acids involved in the glycosylase as well as DNA binding activities of MutY (Wright et al., 1999). Bifunctional glycosylases all bear a conserved lysine residue believed to be important for the initial nucleophilic attack in base removal near their active site, which is lacking in their monofunctional counterparts. To yield more insight into the role of this residue on a structural basis, Williams et al. investigated whether insertion of such a lysine residue into the catalytic site of MutY had any influence on its activity. Indeed, a point-mutation at S120K generated a MutY mutant capable of catalyzing DNA strand scission at a rate that was similar to its A excision activity from A:G and A:8-oxo-G substrates, and also changed it into a bifunctional glycosylase (Williams and David, 2000). This study illustrated that the basic mechanisms of mono- and bifunctional glycosylases were quite similar. The glycosylase activity of MutY was shown to involve a Schiff base intermediate, characteristic for other bifunctional DNA glycosylases that catalyze a β-lyase reaction, though no β-lyase step (per se only performed by bifunctional glycosylases) could be observed (Williams and David, 1998). Reduction of this Schiff-base intermediate with borohydride resulted in the formation of a covalent MutY-DNA adduct. To identify the residues involved in this covalent complex formation, Williams et al. constructed different MutY mutants and identified K142 to be the primary residue for such covalent associations (Williams and David, 1999). As the DNA binding and enzymatic activity of the K142A mutant was comparable to that of the wt enzyme, the formation of this covalent intermediate was not required for removal of A and was suggested to be a consequence of the unusually high affinity of MutY for the product of its glycosylase activity. Similarly, mutation of K142 to glutamine in MutY was shown to also abrogate its ability to form a Schiff base with DNA, while still retaining some of its catalytic activity (Zharkov et al., 2000). Interestingly, this mutation selectively impaired the processing of A:G base pairs, but not of A:8-oxo-G substrates, primarily by interfering with the binding to A:G substrates, but did not impair the catalytic activity per se, again confirming that it was not directly involved in the catalytic step. Using unnatural substrates to elucidate the tolerance of MutY to different modifications of the A or the 8-oxo-G in mismatches in an E. coli-based cellular assay, it was seen that, while modification of A was tolerated rather well, modification of 8-oxo-G resulted in a drastic reduction of base-excision (Livingston et al., 2008). This led to the conclusion that the presence of 8-oxo-G is critical for MutY to recognize A:8-oxo-G mismatches in vivo to initiate repair. D138 and Q37 are both residues that are involved in the catalytic mechanism of MutY-mediated A removal. Interestingly, their substitution yielded mutants with a range of different excision activities. Studies of these mutants demonstrated that changes which reduced the excision activity were better tolerated and less compromising to A:8-oxo-G repair in vivo in E. coli than those affecting the recognition of A:8-oxo-G mismatch affinity (Brinkmeyer et al., 2012). Therefore, this report suggested that the recognition of A:8-oxo-G mismatches was more important for the correct repair of these duplexes than the actual glycosylase activity per se. Interestingly, this can be reconciled with the fact that the release of the substrate by MutY after base-excision is much slower than the actual N-glycosidic activity, seemingly demonstrating that the rate-limiting step of this enzyme is rather the identification of its substrate than the excision step itself. Additionally, this study also revealed which residues are critical for the selectivity and specificity of MutY.

Substrate release. The product release rate of MutY could be greatly enhanced by the two proteins AP-endonuclease IV and exonuclease III, and this effect depended on the presence of the C-terminal domain of MutY (Pope et al., 2002). Also, endonuclease VIII was found to promote MutY dissociation from AP:G substrates, but not from AP:8-oxo-G, and to further process these by βδ elimination (Lu et al., 2006b). This study also showed that MutY interacts with endo VIII through its C-terminus and competes with endo VIII on its natural substrate, the hydroxyurea (hoU):A mismatch, thus inhibiting its activity and possibly reducing the mutagenic effects of hoU. Taken together, it seems important that also the substrate release step is tightly regulated, in order to orchestrate the following steps and to protect the 1-nt gap resulting from base-excision.

The most precise structure of MutY comes from studies with Bacillus stearothemophilus (Lee and Verdine, 2009) (Figure 2). After binding to the 8-oxo-G:A mispair MutY flips out the A from the DNA double-helix. A water molecule is positioned between Asp144 and Asn146 in the MutY lesion-recognition pocket of the enzyme. Earlier studies included biochemical and computational studies on uracil DNA glycosylase (Werner and Stivers, 2000; Dinner et al., 2001) suggested that a so called dissociative action occurs, where the cleavage of the N-glycosylic bond and the subsequent attack of the water molecule on the C1′ (arrow in Figure 2A) do not occur simultaneously, but rather in two discrete steps. In addition Glu43 can adopt a so-called bifurcated hydrogen-bonding interaction of 2.7 and 2.8 Angströms, respectively, with N7 of A (Figures 2A,B). These short distances together with a protonated Glu43, provides acidity and therefore full hydrogen bonding to the N7 of A. As indicated in Figure 2B such a conformation favors the scission of the glycosylic bond between A and the deoxyribose. A similar structure has also been identified for human MUTYH, for which a fragment lacking the first 64 amino-acids was crystalized (Luncsford et al., 2010).

The structure of MutY catalytic core revealed that the two helical domains form a positively-charged groove, positioning the A-binding pocket at their interface (Guan et al., 1998). Also, this study confirmed a nucleotide flipping mechanism by a substitution of the Watson–Crick hydrogen bond partners by protein atoms. Recognition of 8-oxo-G seems to occur independently of double-stranded DNA or of an A-mismatch, and sequential extrusion of 8-oxo-G followed by A occurs in MutY, as demonstrated by Bernards et al. (2002). MutY has been proposed to assemble into a dimer upon substrate binding to yield an active form of the enzyme (Wong et al., 2003). This idea was further substantiated by a study that suggested a model for MutY binding of the mismatched DNA that involves scanning of the DNA by one molecule which enhances binding of second MutY molecule upon encountering an A:8-oxo-G mismatch (Lee et al., 2004).

Kinetically, it has been suggested that the release of A happens fast, while the rate-limiting step was the release of the AP-site (McCann and Berti, 2003). Further investigations into the transition state structure of MutY showed that the irreversible breakage of the N-glycosidic bond could not take place until a H₂O atom was present and that the enzyme stabilized the excision site after excision (McCann and Berti, 2008). Recently, a two-step reaction was proposed to be the basis of the catalytic activity of MutY, as opposed to the three-step mechanism proposed before (Tiwari et al., 2011).

Investigations of the roles of the different H₂O molecules involved in catalysis by MutY from B. stearothermophilus and E. coli suggested that E43 and N7 may be important factors for the activity of MutY (Brunk et al., 2012). Further insight into the roles of the substrate A residues N7 and N3 during catalytic excision by MutY have been gained recently (Michelson et al., 2012).

In E. coli, MutY was shown to be co-transcribed as first gene of a part of a large operon, together with Fpg, the bacterial DNA glycosylase which removes 8-oxo-G from the DNA (Gifford and Wallace, 1999). This further emphasized the involvement of MutY in the repair of 8-oxo-G base pairs in an interplay with Fpg and thus in the response to oxidative DNA damage. Somewhat surprisingly at first glance, oxidative stress down-regulated the activity of MutY by 70% as well as its mRNA levels, and in contrary it was induced more than 2-fold under anaerobic conditions (Yoon et al., 2003). This negative regulation of MutY was mediated by the regulatory genes fur, fnr and arcA. These results were explained with the idea that MutY activity had to be restrained when increased incorporation of 8-oxo-dGTP could possibly take place, which is during times of oxidative stress. This is important, because 8-oxo-dGTP could be inserted opposite a correct templating A, which might erroneously get excised by the action of MutY, thus actually acting promutagenic in this scenario instead of protecting from mutations taking place.

Screening for mutator loci leading to GC→CG transversions in E. coli, Zhang et al. found that inactivation of MutY led to accumulation of these mutations (Zhang et al., 1998). As mentioned above, they showed that MutY bound to G:8-oxo-G mismatches and was capable of removing G from the G:8-oxo-G mispair.

To analyze the impact of mutT, mutM (which encodes the Fpg DNA glycosylase that removes 8-oxo-G from C:8-oxo-G base pairs in bacteria), and mutY on the mutational spectra, following considerations have to be taken into account. In the context of 8-oxo-G and 8-oxo-dGTP (1) CG→AT mutations can arise either from oxidation of C:G to C:8-oxo-G or from incorporation of 8-oxo-dGTP opposite C, followed by wrong incorporation of A opposite 8-oxo-G by the replicative pols during the next round of replication. (2) AT→CG mutations are based on incorporation of 8-oxo-dGTP opposite templating A. Analyzing different combinations of mutated strains in mutT, mutY, and mutM, Fowler et al. found that (1) mutT does not increase CG→AT transversions, regardless of the mutY and mutM background, suggesting that 8-oxo-dGTP does not often get incorporated opposite C but rather opposite A. (2) AT→CG transversions are reduced in mutY and mutMmutY backgrounds, suggesting templating 8-oxo-G preferentially pairs with dATP, which then is a substrate for MutY to excise A from the A:8-oxo-G pair, followed by Fpg that removes 8-oxo-G paired with C. And finally (3) mutY and mutMmutY decrease AT→CG mutations (arising from incorporation of 8-oxo-dGTP opposite templating A) in a mutT wt background, suggesting that a certain amount of 8-oxo-G gets incorporated into DNA even in the presence of functional MutT (Fowler et al., 2003). No strand bias in the mutation rate between leading and lagging strand synthesis in either a mutMmutY or a mutT background could be detected in E. coli using a supF shuttle vector (Watanabe et al., 2001). Interestingly, Bridges et al. showed that the rate of mutation markedly increased in starved mutY mutant E. coli, yielding CG→AT transversion mutations (Bridges et al., 1996). This phenotype could be further enhanced by additional mutation of mutM, even though mutation of mutM alone did not cause this effect. Also, addition of catalase to the plates did not alter the mutation rates, indicating that extracellular H₂O₂ was not involved in the generation of mutations, and it was suggested that singlet oxygen could be the source of internal DNA damage. These findings indicated that MutY may regulate the activity of Fpg in resting cells. Expression of MutY from a mutY-lacZ fusion construct was shown to be enhanced under aerobic compared to anaerobic conditions, but not to be down-regulated by nutrient limitation (Notley-McRobb et al., 2002). However, in many cases, nutrient limitation led to mutY inactivation by deletion, suggesting it might serve as a mechanism to increase mutation rates under these adverse conditions.

Clustered lesions, as induced by ionizing radiation, are defined as two or more lesions formed within one to two helical turns of the DNA. They present a challenge to the repair machinery of the cell. An 8-oxo-G in the vicinity of an AP site was found to retard the processing of the AP site by endo III and Fpg, and the AP site was found to elevate the mutation frequency at 8-oxo-G in wt, nth, fpg, and mutY deleted E. coli (Cunniffe et al., 2007). Interestingly though, the mutation frequency in mutYfpg null cells was reduced by the presence of the AP site, suggesting that the processing of tandem lesions challenges the cellular repair machineries. Similar findings by Noguchi et al. investigating the interplay of 1-nt-gaps and 8-oxo-G lesions in clusters in E. coli demonstrated again, that the mutagenic potential of 8-oxo-G depends on the presence and the position of the gap (Noguchi et al., 2012).

MutY competed with MutS-dependent mismatch-repair when A:C mispairs were present, especially in the presence of an increased dCTP pool (Kim et al., 2003). In E. coli, MutY has been shown to interact via its Fe-S cluster with the ATPase domain of MutS, which enhanced the binding affinity of MutY to A:8-oxo-G mismatches (Bai and Lu, 2007). MutY expression and activity were enhanced in a mutS mutant strain, and AT→GC transversions were reduced by additional mutation of mutY in a mutS background, suggesting a cooperative effect of MutY and MutS in repair of 8-oxo-G damage. Analysis of Bacillus subtilis revealed that the expression of MutY increased drastically upon deletion of mutSL operon in starved cells, possibly to disturb the balance between MutY and MMR proteins to support the production of mutations, which might give growth advantages to these cells (Debora et al., 2011).

In Streptococcus mutans, an oral pathogen, strains with mutations of mutY were shown to display elevated mutation rates, increased resistance to killing by acid and oxidative agents as well as higher virulence compared to the parent strain, suggesting that loss of a BER factor such as MutY could confer an advantage to pathogenic organisms (Gonzalez et al., 2012).

Reconstitution experiments with purified proteins from E. coli revealed, that the presence of Ape1, pol I, and DNA ligase is sufficient to catalyze the entire repair pathway of G:A mismatches in vitro (Au et al., 1989). Further elucidation of the pathway was achieved, when Radicella et al. showed that the average repair tract length initiated by MutY in E. coli is 9–27 nucleotides long, starting at the removed A, and involved pol I, even though the involvement of other pols was also evident (Radicella et al., 1993). This finding was further refined in vitro, when Tsai-Wu et al. found pol I to be responsible for generating these tracts of 5–12 nucleotides length (Tsai-Wu and Lu, 1994).

The substrate specificities for MUTYH are summarized in Table 1. The mammalian homolog of MutY, MUTYH, was first purified from calf thymus and catalyzed removal of A from A:G, A:8-oxo-G and A:C mismatches (McGoldrick et al., 1995). Subsequently, expression and purification of the cloned human protein confirmed its activity to remove A from A:8-oxo-G and A:G base pairs in vitro, supporting that also the human homolog is a bona fide DNA glycosylase (Slupska et al., 1999). Purification of MUTYH from calf liver mitochondria yielded a protein that complexes with A:8-oxo-G, G:8-oxo-G, and T:8-oxo-G, weakly with C:8-oxo-G but not with A:G and A:C mismatches and removed A mispaired with G, C, or 8-oxo-G while weakly removing G from G:8-oxo-G mispairs (Parker et al., 2000). Purification of the murine MUTYH revealed strong similarities to MutY function, even though the intrinsic rates of A removal were lower than by MutY (Pope and David, 2005). Shinmura and colleagues reported that both the purified nuclear and mitochondrial recombinant isoforms of human MUTYH were active, and predominantly removed A from A:8-oxo-G mispairs rather than A:G mispairs under physiological salt concentrations (Shinmura et al., 2000). MUTYH in human cell extracts was shown to be more active in binding and glycosylase activity toward A:G mismatches than recombinant MUTYH expressed in bacteria (Gu and Lu, 2001). Furthermore, the authors found this native form of MUTYH to migrate slower on a non-denaturing polyacrylamide gel than recombinant human MUTYH purified from bacteria. Moreover the native form seems to be phosphorylated, thus apparently enhancing its glycosylase activity predominantly on A:G but also on A:8-oxo-G. As the phosphorylation status of MUTYH did not alter its electric mobility, it was suggested to be possibly associated with other proteins to account for the higher apparent molecular weight. Accordingly, co-migration of APE1 and MUTYH with A:8-oxo-G substrates could be identified. Ohtsubo et al. found that MUTYH likely also harbors an activity to remove 2-OH-A (Ohtsubo et al., 2000). Removal of 2-OH-A from opposite 8-oxo-G or G has been described for murine MUTYH (Pope and David, 2005) and was confirmed for human MUTYH as well (Ushijima et al., 2005). MUTYH from S. pombe was able to remove G from G:8-oxo-G mismatches as efficiently as A from A:8-oxo-G mismatches, and its expression reduced the frequency of GC→CG transversions in an E. coli mutY mutant, suggesting it might be involved in the repair of G:8-oxo-G lesions (Doi et al., 2005).

A:8-oxo-G substrates processed by murine MUTYH were protected from inappropriate access by OGG1 and APE1, thus preventing the formation of DSBs (Tominaga et al., 2004).

A study by Miyako et al. found that mitochondrial DNA (mtDNA) from HeLa cells could be cleaved by recombinant E. coli MutY, in contrast to Fpg which has been shown to barely cleave mtDNA (Driggers et al., 1993; Hegler et al., 1993), and that this cleavage took place roughly at a rate that was expected to correspond to the amount of 8-oxo-G present in endogenous mtDNA (Miyako et al., 2000). Suzuki et al investigated the repair of 8-oxo-G in DNA and 8-oxo-dGTP in 293T cells using supF shuttle plasmids (Suzuki et al., 2010). While knockdown of OGG1, MUTYH, NTH1, and NEIL1 all led to a significant increase in CG→AT transversions caused by the C:8-oxo-G pair in the shuttle plasmid, only knockdown of MUTYH resulted in a reduction in AT→CG transversions induced by 8-oxo-dGTP. In summary, MUTYH displays remarkable similarity to its bacterial homolog MutY regarding its activity and substrate specificity.

The subcellular localization of MUTYH was rather enigmatic for a long time. A study using expression of tagged proteins in COS-7 cells revealed that MUTYH was mainly transported to the mitochondria, which was probably the result of the isoform that was used (Takao et al., 1998). Follow-up work by the same group identified an alternatively spliced transcript differing in exon 1, leading to the nuclear localization of this variant (Takao et al., 1999). Ten further isoforms containing unique 5′sequences that could be grouped into three types were subsequently described, and suggested to encode multiple authentic MUTYH proteins (Ohtsubo et al., 2000). Other reports have further discussed the localization of MUTYH in cells, finding isoforms targeted to the nucleus (Tsai-Wu et al., 2000; Ichinoe et al., 2004) or the mitochondria (Englander et al., 2002; Ichinoe et al., 2004). However, work still needs to be done to analyze the distribution of isoforms to the different subcellular compartments in different cell and tissue types to clarify this matter further.

Analyzing the distribution of endogenous MUTYH in serum-stimulated proliferating MRC5 cells with antibodies, Boldogh et al. reported both nuclear and mitochondrial localization of MUTYH (Boldogh et al., 2001). The nuclear form co-localized with BrdU and the proliferating cell nuclear antigen (PCNA) and, similarly to PCNA, increased 3- to 4-fold to peak during S-phase compared to G1, whereas levels of OGG1 or MTH1 did not change during the cell cycle. These studies suggested a role of targeting MUTYH to the replication fork to ensure that its activity is directed to the newly synthesized template strand. Subsequently, DNA replication was shown to enhance the MUTYH-dependent repair of A:8-oxo-G mismatches in vivo, and it was demonstrated that the interaction with PCNA was critical for the activity of MUTYH (Hayashi et al., 2002). Taken together, these findings clearly support a replication-associated function of MUTYH.

Recently, a number of reports have accumulated that link MUTYH to the DNA damage response and implicate it in apoptotic signaling. To investigate the contribution of nuclear and mitochondrial accumulation of oxidative base lesions to the triggering of apoptosis, Oka and colleagues used OGG1 knockout (ko) cells deficient in the nuclear or mitochondrial form of MUTYH, respectively (Oka et al., 2008). The accumulation of single-strand breaks in nuclear DNA triggered PARP-dependent cell death and could be rescued by depletion of nuclear MUTYH. The same was true for mitochondria, where MUTYH triggered calpain-dependent cell death by single-strand breaks. These results suggested that MUTYH catalyzes the formation of single-strand breaks in both of these DNAs, hence leading to the execution of apoptosis. Exposure of human cells to sodium nitroprusside, an agent that causes 8-oxo-G accumulation in cellular DNA by acting as an NO donor, led to MUTYH-dependent cell death that was initiated by oxidized bases in the mitochondrial, but not the nuclear DNA (Ichikawa et al., 2008). The role of single-strand breaks generated by MUTYH in the induction of cell death was further underlined by the finding that synthetic sickness/lethality mediated by either inhibition of pol β combined with MSH2, a component of the mismatch repair pathway, or pol γ with MLH1, both of which led to a nuclear 8-oxo-G accumulation, could be rescued by silencing of MUTYH (Martin et al., 2010). BER has been implicated in many different pathological conditions of the central nervous system (reviewed in Bosshard et al., 2012). A very recent report implicated MUTYH in degeneration by triggering apoptosis in microglia and neurons through initiation of single-strand breaks during repair of A:8-oxo-G mismatches (Sheng et al., 2012). Nuclear accumulation of 8-oxo-G triggered PARP-dependent apoptosis in microglia, while mitochondrial 8-oxo-G accumulation led to calpain-dependent apoptosis in neurons. All these findings are in agreement with a model, wherein the repair of DNA mismatches by MUTYH leads to generation of toxic single-strand breaks, and thus contributes to cellular death in case of excessive damage burden (i.e., an amount of DNA damage that surpasses the cellular capacity to further process these lesions properly). Thus, this model explains, why under conditions of severe damage the absence of MUTYH is beneficial for the survival of the cells. On the other hand, there are a number of reports that show that loss of MUTYH actually can sensitize cells to DNA damaging agents. Along this line, double mutations in S. pombe MUTYH with RAD1 or, to a lesser extent RAD9, were shown to enhance the sensitivity of the cells to DNA damaging agents and hydroxyurea (Jansson et al., 2008). The consequences of these deficiencies were chromosome segregation defects and checkpoint failure after UV irradiation, as well as morphological defects, even in the absence of DNA damaging agents. This implicated MUTYH in the repair of a wide range of DNA damage and linked it to the checkpoint pathway. Under low-dose oxidative stress, MUTYH OGG1 double-ko mouse cells also showed hypersensitivity to oxidation damage and a reduction of S phase concomitant with an increase of G2/M phase cells, while the levels of cell death remained unchanged (Xie et al., 2008). Furthermore, an increase in centrosome amplifications and formation of multinucleated cells could be observed in the surviving fraction of the ko cells, suggesting an involvement of MUTYH and OGG1 in the regulation of cell-cycle progression and cell division under oxidative stress. Further evidence implicating MUTYH in checkpoint control came from a study showing that siRNA-mediated knockdown of MUTYH resulted in a decreased phosphorylation of ATR and Chk1 upon treatment of HEK293 cells with HU or UV (Hahm et al., 2011). Concomitantly, the authors observed an increase in the phosphorylation of Cdk2 as well as the amount of the Cdc25A phosphatase, suggesting that MUTYH was involved in activation of the DNA damage response.

Thus, there seems to be growing evidence that implicates MUTYH to be an important factor in the cellular response to oxidative stress and inflammatory conditions by an involvement in cell death signaling (as discussed in Oka and Nakabeppu, 2011). Along these lines, MUTYH has been suggested to play a role in mitochondrial dysfunction in the pathogenesis of Parkinson's disease (Fukae et al., 2007; Nakabeppu et al., 2007). However, it still remains to be clarified how MUTYH can initiate apoptosis of cells in some instances, while it seems to protect from apoptosis in others.

The data on cells and mice with biallelic deletion of MUTYH are somewhat discrepant. MUTYH ko embryonic stem cells displayed a mutator phenotype, but did not show any hypersensitivity toward oxidative stress induced by H₂O₂ or menadione (Hirano et al., 2003). A study with mutyh^−/− knockout mice by Xie et al revealed no significant increase in survival or tumor incidence after 14 months, suggesting that MUTYH deficiency is not sufficient to cause a tumor-predisposition (Xie et al., 2004). This study also showed that combined ko of MUTYH with OGG1 led to a decrease in life span and increased tumor formation for double ko mice compared to single ko. Interestingly, 75% of the lung tumors showed an activating GC→TA transversion mutation at codon 12 of K-ras, a feature that is often detected in MUTYH-associated polyposis (MAP) tumors, but none in the p53 gene or in the adjacent normal tissues. Additional heterozygosity for Msh2 (mutyh^−/− ogg1^−/− msh2^±) did not inflict on the total tumor incidence but accelerated malignant lung and ovarian tumor formation in the mutyh^−/− ogg1^−/− background. A complete knockout of Msh2 to generate triple ko (mutyh^−/− ogg1^−/− msh2^−/−) further increased tumor incidence and decreased survival time, but did not differ from the phenotype displayed by msh2^−/− single knockouts. This was suggested to be due to the strong mutator phenotype of msh2^−/− mice that might mask additional difference caused by mutyh^−/− and ogg1^−/−.

Spontaneous mutagenesis in the small intestine of ogg1^−/− and mutyh^−/− ogg1^−/− double deficient mice at the age of 4–5 weeks using a transgene reporter revealed increased mutations in the double-ko's but not in the ogg1^−/− mice (Isogawa, 2004). Furthermore, the GC→TA mutation frequency increased in mutyh^−/− and in ogg1^−/−and a cooperative increase could be observed in mutyh^−/− ogg1^−/−, suggesting a cooperative function between OGG1 and MUTYH to prevent 8-oxoG-related mutagenesis. Russo et al. also reported an additive effect in mutyh^−/− ogg1^−/− on the age-dependent increase in 8-oxo-G levels in lung and small intestine compared to the single ko's (Russo et al., 2004). Strikingly, these tissues were identical with the ones that showed increased cancer incidence in mutyh^−/− ogg1^−/− mice in the study by Xie et al. (2004). MUTYH deficiency in a background of APC^min/+ mice led to the occurrence of stop-codons in the APC gene by induction of CG→AT transversion mutations and thus promoted intestinal tumorigenesis (Sieber et al., 2004).

In 2007 a study reported an increased susceptibility to spontaneous and stress-induced tumorigenesis in a large cohort of mutyh^−/− mice kept for 18 months, strongly contradicting data on mutyh^−/− obtained by different groups thus far (Sakamoto et al., 2007). This suggested that presence of a MUTYH deficiency is sufficient to predispose for malignancies of the intestinal tract, such as lymphoma and adenoma. More impressively still, oral KBrO₃ treatment of mutyh^−/− mice led to a dramatic increase in CG→AT transversion mutations and small intestinal tumors. The authors claimed that the tumor-prone phenotype might have been missed earlier due to genetic differences in the mouse strains and the older age at which the tumor burden was evaluated in their study. This was in line with the fact that many of the studies with mutyh^−/− mice have been reporting a strong tendency toward age-dependent accumulation of 8-oxo-G in tissues. In general, in light of the huge complexity of the disease, it can be debated, whether mice are useful cancer models to compare with the human disease, due to the entirely different life span, metabolism, inbreeding status and many other aspects.

As noted above, the combination mutyh^−/− and msh2^−/− did not greatly affect the mutation rate. However, the loss of mutyh^−/− combined with msh2^−/− significantly increased the amount of oxidative DNA damage in several organs compared to msh2^−/− mice, suggesting an independent contribution of both genes to genetic maintenance (Russo et al., 2009). Surprisingly, the development of metastasizing lymphoma and the time of death were significantly delayed in the mutyh^−/−msh2^−/− mice compared to msh2^−/−, suggesting that the cancer-prone phenotype of the double knockouts depends substantially on the activity of MUTYH (Russo et al., 2009). The relationship of MUTYH and MMR is reviewed in more detail in Russo et al. (2007).

In a mouse model of ulcerative colitis MUTYH was shown to play a major role in propagating the inflammatory response that lead to the onset of chronic colitis (Casorelli et al., 2010). Taken together, all the data analyzing the function of MUTYH in vivo strongly supports the idea that MUTYH-mediated correction of A:8-oxo-G mispairs plays an important role in the maintenance of genetic integrity and protects cells from malignant transformation.