Vol. 54 No. 3/2007, 435–457 Review

Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.


INTRoDuCTIoN
Maintaining low mutation rates is essential for the cell stability.However, natural isolates of Escherichia coli have been found to have elevated mutation rates (Matic et al., 1997) and strains showing this phenotype are termed mutators.Although the mutator phenotype may have some beneficial effects allowing better adaptation to environmental conditions, it also generates many deleterious and lethal mutations (Funchain et al., 2000).
The first described E. coli mutator gene -mutT1 (Treffers et al., 1954) which specifically increases, from 100 to 10 000-fold, the occurrence of AT→CG transversions (Yanofsky et al., 1966) was shown to encode MutT pyrophosphohydrolase specifically acting on 8-oxodGTP (Maki & Sekiguchi, 1992), thus preventing incorporation of this po-2007 K.D. Arczewska and J.T. Kuśmierek tentially mutagenic substrate into DNA.The list of E. coli mutators was extended further by other DNA repair gene products, such as the base excision repair (BER) glycosylases MutM/Fpg and MutY (for details see Krwawicz et al., this issue), mismatch repair (MMR) proteins MutH, MutS and MutL (for details see below), and MutU/UvrD -helicase II engaged in MMR, nucleotide excision repair (NER) (Truglio et al., 2006; for details see Maddukuri et al., this issue), and recombination repair (RR) (for details see Vidakovic et al., 2005;O'Driscoll & Jeggo, 2006;Nowosielska, this issue).Additionally, Miller (1996) extended the list of E. coli mutators by ung, sodA, dam, oxyR, and polA strains defective in uracil-DNA glycosylase, superoxide dismutase, DNA adenine methyltransferase, positive regulator of oxidative damage response, and DNA polymerase I, respectively, but all of them are rather weak mutators and thus are not considered as major E. coli mutators.The list of E. coli mutators is not limited to strains defective in DNA repair, but also includes strains encoding mutated tRNAs, such as mutA and mutC (Slupska et al., 1996), and mutated 3'→5' proofreading ε subunit of the DNA polymerase III holoenzyme -mutD/dnaQ (Echols et al., 1983), but they are not subject of this review.
Counterparts of bacterial DNA repair proteins have been found in eukaryotic organisms, including humans.Moreover, it has been shown that DNA repair deficiency results in accumulation of DNA damage, which may contribute to aging and development of human diseases, including cancer and neurological diseases (Brooks, 2002;Krokan et al., 2004;Olinski et al., 2007).The present review describes two DNA repair and damage prevention systems -nucleotide pool sanitization and mismatch repair.For an overwiev of the human repair proteins described below see Table 1.

NuCleoTIDe Pool DAMAGe AS SouRCe of MuTATIoNS AND ITS PReveNTIoN bY E. coli MutT PRoTeIN
Various DNA damaging agents react with nucleic acid bases present not only in DNA (for a review see Krwawicz et al., this issue), but also in precursors for DNA synthesis, i.e. 2'-deoxyribonucleoside-5'-triphosphates (dNTPs).Generally, bases in dNTPs are more easily accessible to damage than bases in DNA, where they are involved in secondary and tertiary DNA as well as chromosomal structures (Topal & Baker, 1982;Kamiya & Kasai, 1995).Modified dNTPs may induce mutations, since they are incorporated into DNA by DNA polymerases with an efficiency within the range of 10 -5 -10 -2 of unmodified dNTPs incorporation (for examples see: Snow et al., 1984;Purmal et al., 1994;Miller et al., 2000;Imoto et al., 2006).In fact, one of the most common oxidative modifications in the dNTP pool -8-oxodGTP -has been shown to be incorporated almost 24 times more efficiently opposite template A than opposite template C by human polymerase β (Miller et al., 2000).Thus, 8-oxodGTP misincorporated opposite A may lead to AT→CG transversions (for details see Fig. 1) both in vitro (Pavlov et al., 1994;Minnick et al., 1994) and in vivo (Inoue et al., 1998;Satou et al., 2005).
To prevent 8-oxodGTP incorporation to DNA, E. coli cells are equipped with the MutT protein, which was discovered as a nucleoside triphosphate pyrophosphohydrolase dephosphorylating all canonical ribo-and 2'-deoxyribonucleoside-5'-triphosphates to their corresponding 5'-monophosphates and inorganic pyrophosphate (dNTP + H 2 O → dNMP + PPi) (Bhatnagar & Bessman, 1988;Bhatnagar et al., 1991).Initially, the MutT protein was proposed to prevent the occurrence of AT→CG transversions by degrading a specific form of dGTP, or dGTP in the syn conformation, which can mispair with A during replication (Akiyama et al., 1989;Bhatnagar et al., 1991).However, the discovery that the MutT protein is over 2000 times more active towards 8-oxodGTP than towards dGTP, has pointed out to its true role (Maki & Sekiguchi, 1992).Recently it appeared that the MutT protein is also able to efficiently hydrolyze 8-oxodGDP (Ito et al., 2005).8-OxodGTP and 8-oxo-dGDP are interconvertible, probably by the actions of nucleoside diphosphate kinase (NDPK) and nucleoside triphosphatase (Hayakawa et al., 1995;Kamiya & Kasai, 1999).Furthermore, MutT protein also prevents transcriptional errors by dephosphorylation of ribonucleotides 8-oxoGDP and 8-oxoGTP, which otherwise can be incorporated into RNA opposite A present in the DNA template (Taddei et al., 1997;Ito et al., 2005).
A comparison of the amino-acid sequence of the MutT protein with sequences present in databases has revealed similarities with putative products of uncharacterized open reading frames (Orfs) from bacteria to mammals, and also with viral gene products of unknown function.All these similarities were concentrated in the same MutT segment consisting of about 30 amino acids, with six positions containing strictly conserved amino-acid residues.Based on this sequence homology the MutT protein family was distinguished as a family of proteins containing the MutT signature sequence, i.e.Gx 5 Ex 7 REUxEEx 2 U (where x means any residue and U means a bulky aliphatic or hydrophobic residue, i.e.I, L, V, M, F, Y or W) (Koonin, 1993).In consequence, functional MutT homologues from Proteus vulgaris and Streptococcus pneumoniae were identified, characterized, and shown to complement the mutator phenotype of the Nucleotide pool sanitization and mismatch repair systems mutT E. coli strain (Kamath & Yanofsky, 1993;Bullions et al., 1994;Mejean et al., 1994).Furthermore, human, rat and mouse MutT homologue-1 (MTH1) genes have been identified, cloned and shown to suppress the increased occurrence of AT→CG transversions in E. coli mutT cells (Mo et al., 1992;Sakumi et al., 1993;Furuichi et al., 1994;Cai et al., 1995;Kakuma et al., 1995).Despite the functional homology, human MTH1 and E. coli MutT share only 30 residues (23%), 14 of which are contained in the conserved 23-residue module, while the other 16 residues are scattered throughout the whole molecules (Shimokawa et al., 2000).
The MutT protein family appeared to contain also proteins active in many other reactions, distinct from the MutT-like activity, including hydrolysis of nucleoside-5'-di-and triphosphates, dinucleoside and diphosphoinositol polyphosphates, nucleotide sugars and alcohols, dinucleotide coenzymes and RNA caps (Bessman et al., 1996;McLennan, 2006).In all the cases where the enzymatic function was known these proteins appeared to be pyrophosphohydrolases that acted upon a nucleoside diphosphate linked to some other moiety, X, hence the name "Nudix" hydrolases was proposed for this family, with the term "MutT signature sequence" changed to "Nudix box".MutT and its functional homologues constitute a subfamily of Nudix hydrolases, where X = phosphate group.It has been proposed that Nudix hydrolases are "housecleaning" enzymes which control the level of cellular metabolism by-products, metabolic intermediates and signaling compounds, whereas the specific role of MutT proteins is to "sanitize" the dNTP pool (Bessman et al., 1996).
The importance of nucleotide pool sanitization is further highlighted by the observation that dUT-Pase, enzyme responsible for elimination of another damaged dNTP, dUTP, is essential for survival of E. coli (el-Hajj et al., 1988), S. cerevisiae (Gadsden et al., 1993;Guillet et al., 2006) and C. elegans (Dengg et al., 2006).Interestingly, it was shown recently that abrogation of the S-phase checkpoint gene clk-2 rescued  Michaels and Miller, 1992).8-OxoG is formed in DNA both by direct guanine oxidation and by 8-oxodGTP incorporation from nucleotide pool.8-OxodGTP is incorporated mainly opposite A, and thus, if unrepaired may lead to AT→CG transversion.When 8-oxoG is present in DNA it may pair with A upon replication, which leads to GC→TA transversions.hMTH1 pyrophosphohydrolase, and hOGG1, hOGG2 and hMYH glycosylases act together to prevent these mutations.lethality and developmental defects, and eliminated cell cycle arrest and apoptosis induced by dUTPasedepletion in C. elegans.Therefore, it appears that dUMP misincorporation to DNA leads to checkpoint activation after processing by uracil-DNA glycosylase, and abrogation of the CLK-2 checkpoint leads to tolerance of DNA-repair intermediates (Dengg et al., 2006).Furthermore, methylated nucleotides, such as 1-medATP, may be repaired by E. coli oxidative demethylase AlkB (Koivisto et al., 2003); for a review of AlkB protein see Nieminuszczy & Grzesiuk (this issue).
Human MutT homologue (hMTH1) gene spans 9 kb, is localized on chromosome 7p22 (Furuichi et al., 1994), and consists of five major exons, with exon 1 consisting of two segments (1a and 1b), exon 2 consisting of three segments (2a, 2b and 2c), and exons 3, 4 and 5 without segmentation.Alternative splicing results in formation of seven types of transcripts (1, 2A, 2B, 3A, 3B, 4A and 4B), with type 1 mRNA transcript predominating in most or all human cells and tissues (Oda et al., 1997).All transcripts direct formation of a 156-amino-acid (18-kDa) hMTH1 protein isoform (termed p18) from the same AUG4 located at the beginning of exon 3. Additionally, B type mRNAs (2B, 3B and 4B) have three additional upstream AUGs (AUG1, AUG2 and AUG3) localized in-frame with AUG4.AUG1 is followed by a termination codon, so functional products are produced only from AUG2, AUG3 and AUG4.Therefore, B-type mRNAs produce additionally a 171-amino-acid (p21) and a 179-amino-acid (p22) polypeptide from AUG3 and AUG2, respectively.Western blot analysis of Jurkat and HeLa cells crude extracts revealed the existence of all three isoforms of MTH1 protein (i.e.p18, p21 and p22), with the p18 isoform constituting 90%.Additionally, a single nucleotide polymorphism (SNP) is present at the 5' splice site (GT→GC) of exon 2c segment, which alters the splicing pattern of exon 2c.This polymorphism destroys the termination codon after AUG1, which generates an extended open reading frame coding for a 197-amino-acid polypeptide (p26) (Oda et al., 1997).The frequency of the C allele was estimated at about 7-9% in Japanese population (Kohno et al., 2006).Computer modeling revealed that p18 and p26 proteins contain a mitochondrial targeting sequence, and the additional N-terminal 18-aminoacid fragment of the p26 isoform constitutes a better mitochondria-targeting signal than that found in p18 isoform.All four hMTH1 isoforms were shown to have enzymatic activity (Sakai et al., 2006).One more polymorphism was discovered in exon 4 in codon 83 of the p18 hMTH1 protein coding sequence, where GTG encoding valine is changed to ATG encoding methionine (Wu et al., 1995).The Met83 variant was shown to be more thermolabile, more hydrophobic, have a higher α-helix content and lower catalytic activity than Val83 (Yakushiji et al., 1997).The frequency of this type of polymorphic alteration in the hMTH1 allele was estimated at about 9% in the Japanese population.There is a tight linkage between the two hMTH1 polymorphic sites, Met83 and GC at exon 2c, or Val83 and GT at exon 2c, which results in the synthesis of Met83-hMTH1 (p26), but not Val83-hMTH1 (p26) (Oda et al., 1999).Other polymorphisms reported for hMTH1 are as follows: T to C at codon 45 in exon 3, with C allele frequency 2.33%, silent C to T polymorphism at codon 119 in exon 5, with T allele frequency 2.03%, C to T polymorphism in intron 3, and G to A polymorphism at position 92, resulting in the Arg31Gln change (Wu et al., 1995;Sieber et al., 2003;Jiang et al., 2005).

Role of hMTH1 IN HuMAN DISeASe
It has been proposed that an early step in carcinogenesis is elevation of the rate of spontaneous mutations, i.e. development of a mutator phenotype (Loeb, 2001;Beckman & Loeb, 2006;Venkatesan et al., 2006).Since MutT-deficient E. coli cells show a clear mutator phenotype, the hMTH1 gene was suspected to be one of the genes whose deficiency would be involved in cancer progression.Consistently with this assumption, MTH1-knockout mice showed a higher incidence of lung, liver and stomach cancer (Tsuzuki et al., 2001a;2001b).However, no mutations or polymorphisms in the hMTH1 gene were found to be correlated with hereditary nonpolyposis colorectal cancer (HNPCC) (Wu et al., 1995), acute childhood leukemia (Lin et al., 1998b), hepatocellular carcinoma, lung cancer (Oda et al., 1999), ovarian cancer (Takama et al., 2000), familial adenomatous polyposis (FAP), sporadic colorectal cancer (Sieber et al., 2003;Kim et al., 2004), nor with Parkinson's disease (Satoh & Kuroda, 2000).Similarly, no such correlation was found in the rat 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)induced mammary carcinomas, which showed an elevated level of AT→CG transversions (Okochi et al., 2002a;2002b).On the other hand, the Val83Met polymorphism is suggested to be involved in the development of type 1 diabetes mellitus in female Japanese (Miyako et al., 2004), and together with the T/C polymorphism in exon 2, with the risk of small cell lung cancer (SCLC), but not with non-small cell lung cancer (NSCLC) (Kohno et al., 2006).Furthermore, the Val83Met polymorphism was shown to be more frequent in gastric cancer patients, and the Met83 variant correlated with somatic mutations in TP53 tumor suppressor gene (Kimura et al., 2004).
Surprisingly, in various types of tumors and disease states, and in rodent models of human diseases, MTH1 overexpression was found to be more common than its mutation.Thus, MTH1 mRNA was shown to be overexpressed in renal cell carcinoma (Okamoto et al., 1996), lung cancer cells and NSCLC tissues (Hibi et al., 1998;Kennedy et al., 1998), hepatocellular carcinoma (Zhou et al., 2005), breast cancer (Wani et al., 1998), PhIP-induced rat mammary carcinomas (Okochi et al., 2002a;2002b), MTH1 protein level was shown to be increased in brain tumors (Iida et al., 2001), NSCLC (Kennedy et al., 2003), colorectal cancer (Koketsu et al., 2004), lung epithelial cells of patients with idiopathic interstitial pneumonias (Kuwano et al., 2003), mouse heart after myocardial infarction (Tsutsui et al., 2001), and hMTH1 activity was shown to be increased in NSCLC (Speina et al., 2005) in comparison with non affected tissues or cells.Furthermore, hMTH1 overexpression was also observed in regions involved in oxidative stress-in-duced damage in brains of patients with Parkinson's (Shimura-Miura et al., 1999) and Alzheimer's disease (Furuta et al., 2001), in nuclei of motor neurons of patients with amyotrophic lateral sclerosis (Kikuchi et al., 2002), and also was shown to protect mouse neurons from oxidative stress damage in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced Parkinson's disease model (Yamaguchi et al., 2006), and in kainate-induced excitotoxicity (Kajitani et al., 2006).Furthermore, MTH1 was also shown to suppress H 2 O 2 -induced cell death in mouse embryo fibroblasts (Yoshimura et al., 2003).Consistently with the above results, MTH1 overexpression was observed under oxidative stress induced by H 2 O 2 in cultured glioma cells (Iida et al., 2004), human skin fibroblasts and Jurkat cells (Meyer et al., 2000), and in cells exposed to various toxic agents, such as in the case of human lung tissues of tobacco-smoking NSCLC patients (Arczewska et al., in preparation), human fibroblasts exposed to ionizing radiation (Haghdoost et al., 2006), in livers of rats treated with carbon tetrachloride (Takahashi et al., 1998), rat lung epithelial cells treated with urban particulate matter (Choi et al., 2004), human lung epithelial cells treated with crocidolite asbestos (Kim et al., 2001), and also in tissues exposed to a high level of toxic metabolites excreted from the organism, such as the rat kidney inner cortex (Kasprzak et al., 2001) and human colorectal cancers located in the distal part of the colon (Koketsu et al., 2004).Furthermore, hMTH1 overexpression was observed under increased oxygen consumption, i.e. in leukocytes of healthy subjects after exercises (Sato et al., 2003).All the above observations have led to the conclusion that hMTH1 overexpression is a molecular marker of oxidative stress, especially in cancer cells (Kennedy et al., 1998), and was even proposed to be a marker for diagnosis of patients with non-small cell lung cancer (Chong et al., 2006).In fact, hMTH1 overexpression has proved to be a reliable marker of oxidative stress in cancer and other diseases, since a high level of its expression in peripheral lymphocytes was shown to be associated with increased risk of prostate cancer (Liu et al., 2003), and its overexpression was observed in lymphocytes of uremic patients (Tarng et al., 2004).
The role of MTH1 in oxidative-damage prevention is further highlighted by the observation that hMTH1 mRNA level is inversely correlated with 8-oxoG DNA level in human lung cancer cell lines (Kennedy et al., 1998) and in leukocytes of healthy subjects after exercise (Sato et al., 2003), a higher hMTH1 mRNA level coincides with lower 8-oxoG DNA levels in human lung epithelial cells treated with crocidolite asbestos (Kim et al., 2001), a higher MTH1 activity coincides with lower 8-oxoG DNA levels in fetal compared to maternal mouse organs (Bialkowski et al., 1999b), a higher hMTH1 Nucleotide pool sanitization and mismatch repair systems protein level coincides with higher 8-oxodGuo levels in the cytoplasm and mitochondria of substantia nigra neurons of patients with Parkinson's disease (Shimura-Miura et al., 1999) and the hMTH1 protein level is positively correlated with extracellular 8-oxodGuo level in cell cultures (Haghdoost et al., 2006).Furthermore, treatment of rats with cadmium (II) (Cd(II)), which inhibits the activity of the MutT and MTH1 proteins (Porter et al., 1997;Bialkowski & Kasprzak, 1998), resulted in a decrease of MTH1 activity concurrently with an increase of 8-oxoG level in DNA of the testis, the target organ of Cd(II)-induced mutagenesis (Bialkowski et al., 1999a).
Interestingly, although MTH1 expression was found to be increased in replicating cells (Wani & D'Ambrosio, 1995), stimulated by phytohemagglutinin and interleukin-2 (Oda et al., 1997), and higher in tissues with highly proliferating cells, such as thymus and testis, than in tissues with non-proliferating cells, such as brain (Kakuma et al., 1995;Igarashi et al., 1997;Oda et al., 1997), Bialkowski and Kasprzak (2004) have shown that MTH1 protein activity is not regulated by the cell proliferation rate.Furthermore, the MTH1 protein activity does not depend on the cell cycle stage, and is not changed under serum starvation of cultured cells, but decreases with increasing cell population density (Bialkowski & Kasprzak, 2000).Therefore, although MTH1 overexpression under oxidative stress and in cancer cells is a well-recognized feature, the actual mechanisms that are involved in this phenomenon remain to be elucidated.

PReveNTIoN of 8-oxoG-INDuCeD MuTATIoNS bY Go SYSTeM -CooPeRATIoN of THe hMTH1 PRoTeIN AND beR PATHwAY
8-OxoG may be formed in DNA by G oxidation or by 8-oxodGTP incorporation opposite A or C (see Fig. 2).In E. coli 8-oxoG paired with C is removed by MutM protein, but, if unrepaired it may pair with A upon replication and thus GC→ TA transversions occur.On the other hand, A is removed from the 8-oxoG•A pair by MutY protein, which prevents GC→TA transversions.Paradoxically, if 8-oxoG comes from 8-oxodGTP incorporated opposite A, then removal of A by MutY would induce AT→CG transversions, since 8-oxoG may pair with C upon replication.Therefore, the MutT protein hydrolyzing 8-oxodGTP is crucial in prevention of AT→CG transversions.Altogether, MutM, MutY and MutT were proposed to cooperate in prevention of 8-oxoG (GO)-induced mutations, and this prevention system was termed GO (Michaels & Miller, 1992).Consistently, GC→TA transitions are greatly increased in an E. coli mutMmutY double mutant, but in the triple mutant mutMmutYmutT their frequency is not further increased.This phenomenon may be explained by 8-oxoG removal by Nei glycosylase (for a review see Krwawicz et al., this issue) or other DNA repair systems, such as MMR (see below) or NER (Czeczot et al., 1991;Bregeon et al., 2003).On the other hand, mutMmutYmutT and mutYmutT show a lower level of AT→CG transversions than mutMmutT and mutT, which confirms that MutY activity in fact enhances 8-oxodGTP-induced mutagenesis (Fowler et al., 2003).
Mammalian cells possess three main N-glycosylases that prevent 8-oxoG-induced mutations: OGG1, which preferentially removes 8-oxoG from pairs with C or T, OGG2, which removes 8-oxoG paired with G or A, and MYH, which removes A from the pair with 8-oxoG.Mammalian OGG2 has been proposed to remove mainly 8-oxoG incorporated from the cellular nucleotide pool (for a review see (Nakabeppu et al., 2006)).Similarly like in E. coli cells, OGG1 and MYH are key players in GC→TA transversions prevention, and OGG1 −/− MYH −/− double knockout mice show an increased level of G→ T transversions together with a very high incidence of tumors (Xie et al., 2004).Surprisingly, MTH1 disruption appeared to suppress lung tumorigenesis in OGG1-knockout mice, which was attributed to the increased cell death of damaged tumor progenitor cells upon extensive 8-oxoG incorporation in DNA and RNA (Sakumi et al., 2003).

MISMATCH RePAIR (MMR)
Mismatch repair (MMR) is the major postreplicative DNA repair system, which increases replication fidelity up to 1000-fold (Modrich & Lahue, 1996;Schofield & Hsieh, 2003).MMR removes primary replication errors that escaped DNA polymerase proofreading, such as base-base mismatches and small insertion/deletion loops (IDLs), which are most easily formed in long repetitive sequences, i.e. in microsatellites.Thus, defects in MMR induce the mutator phenotype characterized by changes in the microsatellites length, termed microsatellite instability (MSI).MSI is an established biomarker for MMR dysfunction in tumor cells (Umar et al., 2004).

Methyl-directed MMR in E. coli
Key players in the E. coli MMR system, MutS, MutL, MutH and UvrD were identified in studies of mutator strains (Cox et al., 1972;Wagner & Meselson, 1976), and the whole system was reconstituted in vitro (Lahue et al., 1989).MMR preferentially repairs the newly synthesized strand, and in E. coli strand discrimination is based on the fact that adenine is methylated in GATC sequences by Dam methyltransferase about 2 min after DNA synthesis, therefore the newly synthesized strand is transiently unmethylated (Lyons & Schendel, 1984).Consistently with its role in MMR, dam E. coli cells are weak mutators (Glickman, 1979).
Initially, MutS protein dimer (or tetramer) recognizes and binds IDLs containing up to about four unpaired bases (Parker & Marinus, 1992), and also seven of eight possible mismatches (Su & Modrich, 1986;Su et al., 1988).MutS binding affinities and mismatch repair efficiencies vary with the composition of the mismatch and local sequence context, with G•T and C•A mismatches being preferentially repaired in most of the tested systems (Kramer et al., 1984;Dohet et al., 1985;Jones et al., 1987;Brown et al., 2001).Consistently, defects in MMR genes induce mainly GC→AT and AT→GC transitions, and frameshift mutations (Lahue et al., 1989).The C•C mismatch is almost not recognized by MutS and it was postulated to be repaired by an MMR-independent pathway (Nakahara et al., 2000).Further, mismatch-bound MutS recruits MutL dimer in an ATP-dependent manner (Grilley et al., 1989).The MutL protein is an ATPase and is thought to be a "molecular matchmaker" which mediates the interaction between MutS and MutH (Modrich, 1991).Thus formed, the ternary complex of MutS(ATP)-MutL-mismatch activates monomeric MutH endonuclease which incises an unmethylated GATC sequence at a site 5' or 3' to the mismatch, located even 1000 bp from the mismatch (Welsh et al., 1987;Bruni et al., 1988).The resulting nick serves as the point of entry for MutL-activated UvrD helicase, which unwinds DNA double helix from the nick to about 100 nucleotides past the mismatch, and single-stranded DNA binding (SSB) protein, which stabilizes the single-stranded gap (Lahue et al., 1989).After unwinding the ssDNA flap is degraded in the 5'→3' direction by ExoVII or RecJ exonuclease, if the incision occurred 5' to the mismatch, or in the 3'→ 5' direction by ExoI, ExoVII or Exo X exonuclease, if the incision occurred 3' to the mismatch (Cooper et al., 1993;Grilley et al., 1993;Burdett et al., 2001).Finally, the SSB-stabilized single-stranded gap is filled in by DNA polymerase III holoenzyme and DNA ends are sealed by LigI.Importantly, β clamp, which is a polymerase processivity factor, and γ complex, which loads β clamp onto the DNA helix are required for MMR in vitro (Lahue et al., 1989), and β clamp was shown to interact with MutS (Lopez de Saro & O'Donnell, 2001).
Although MutS and MutL proteins are evolutionarily conserved, the MutH endonuclease is restricted only to Gram-negative bacteria (Jiricny, 2006).Thus, in eukaryotic cells the signals that direct MMR to the newly synthesized strand remain uncertain.Initially it has been proposed that strands are discriminated on the basis of cytosine methylation, analogously to the role of adenine methylation in E. coli cells, but this hypothesis has not been veri-Nucleotide pool sanitization mismatch repair systems fied (Drummond & Bellacosa, 2001;Petranovic et al., 2000).More plausible hypotheses suggest that natural single-strand breaks, occurring as replication intermediates, the replication complex, especially proliferating cell nuclear antigen (PCNA), or proteins segregating with individual strands after replication may be involved.This was supported by the observation that mutH E. coli strains are able to carry out MutHindependent MMR, both in vivo and in vitro, from a single-strand break located at the vicinity of the mismatch (Lahue et al., 1989;Kramer et al., 1984;Bruni et al., 1988).The same was observed in human in vitro MMR assays (Holmes et al., 1990;Thomas et al., 1991;Iams et al., 2002).Furthermore, similarly as observed in E. coli, the eukaryotic β clamp counterpart -PCNA -interacts with yeast and human MutS and MutL homologues (Umar et al., 1996;Clark et al., 2000;Flores-Rozas et al., 2000;Kleczkowska et al., 2001), and mutations in PCNA that abolish interaction with MSH3 and MSH6 confer partial mutator phenotype in vivo (Johnson et al., 1996;Chen et al., 1999;Clark et al., 2000;Flores-Rozas et al., 2000;Lau et al., 2002;Lau & Kolodner, 2003).Thus, PCNA is implicated not only in gap filling repair synthesis, but also in early stages of MMR (Umar et al., 1996;Gu et al., 1998).Moreover, PCNA and eukaryotic clamp loader replication factor C (RFC) were shown to be essential for bi-directional excision during MMR (Dzantiev et al., 2004).Human MSH2 and MSH3 interact also with MMR exonuclease -Exo1 (Schmutte et al., 1998;2001;Rasmussen et al., 2000).
Final steps of MMR include mismatch excision and DNA resynthesis.When a single-strand break is localized at the 5' side of the mismatch, Exo1, stimulated by MutSα hydrolyzes DNA in the 5'→3' direction in an ATP-, mismatch-, and replication protein A (RPA)-dependent manner (Lin et al., 1998a;Genschel et al., 2002;Lee Bi et al., 2002).RPA plays a role similar to E. coli SSB, since it protects ssDNA from incision by nucleases (Ramilo et al., 2002).Furthermore, eukaryotic MMR is apparently helicase-independent (Bennett et al., 1997;Langland et al., 2001;Pedrazzi et al., 2001), and thus RPA binding has been proposed to play some role in DNA unwinding.Exo1 is the only eukaryotic MMR exonuclease, and Exo1-deficient mice are prone to lymphomas and exhibit MSI, as well as the mutator phenotype (Wei et al., 2003;Tran et al., 2004).The single stranded gap is stabilized by RPA.RPA reduces Exo1 processivity by binding ssDNA, and Exo1 is further inhibited by MutSα and MutLα upon reaching the mismatch.This leads to termination of excision.In consequence (A) In the molecular-switch model (Gradia et al., 1999) MutSα normally exists in ADP-bound form, and upon mismatch binding ADP is exchanged to ATP, which induces conformational changes and ATP-independent diffusion of multiple MutSα (possibly in complex with MutLα) sliding clamps along DNA helix.(B) In the active-translocation model (Blackwell et al., 1998;Martik et al., 2004), MutSα clamp (possibly in complex with MutLα) translocates along DNA and ATP is used as energy source to drive its motion.(C) In the DNA bending/verification model (Wang & Hays, 2003;2004) MutSα remains at the mismatch and makes contact with strand break through DNA bending.MutS is able to sense the mismatch, and when bound to DNA without mismatch it hydrolyzes ATP and in consequence becomes displaced from the DNA.On the other hand, when MutS binds DNA containing mismatch, ATP is not hydrolyzed, what leads to activation of downstream MMR effectors.(D) When MutSα-MutLα complex comes into contact with PCNA and RFC bound to strand break, it triggers further MMR steps.MutSα, PCNA and RFC activate latent MutLα endonuclease (Kadyrov et al., 2006), which cleaves DNA at both sides of the mismatch in ATP-dependent manner.Next, at the 5'-break (created by MutLα endonucleolytic cleavage or preexisting in DNA) MutSα-MutLα complex displaces RFC from complex with PCNA and loads Exo1, which hydrolyzes DNA in 5'→3' direction.Nucleotide pool sanitization and mismatch repair systems excision terminates at about 100 nucleotides the mismatch (Genschel & Modrich, 2003;Nielsen et al., 2004).However, Exo1 lacks the 3'→5' exonuclease activity, and a complex consisting of MutSα, MutLα, PCNA, Exo1, and RFC is essential for MMR from a single-strand break located at the 3' side of the mismatch (Dzantiev et al., 2004;Guo et al., 2004).RFC has been proposed to play a double role in 3'directed excision: it loads the PCNA clamp, but also suppresses 5'→3' excision by Exo1 from a singlestrand break located 3' to the mismatch (Dzantiev et al., 2004).It has been proposed that 3'→5' excision is mediated by Pol δ or Pol ε proofreading activity, which was supported by genetic and biochemical inhibition studies conducted in vivo in S. cerevisiae and in vitro with HeLa extracts (Tran et al., 1999;Wang & Hays, 2002).The recent discovery that MutLα has an endonuclease activity, stimulated by MMR cofactors (MutSα, MutLα, PCNA, RFC, ATP and divalent cations) (Kadyrov et al., 2006) may explain the MMR mechanism in a situation when the single strand break is located at the 3' side of the mismatch.Thus, the 5' nick introduced by this endonuclease activity specifically in the discontinuous strand may serve for Exo1 degradation in substrates containing a break localized 3' to the mismatch.Finally, DNA polymerase δ fills the gap, in the presence of PCNA (Gu et al., 1998) and RPA (Lin et al., 1998a;Ramilo et al., 2002), and finally DNA ligase (probably LIG1) seals the ends (Constantin et al., 2005;Zhang et al., 2005).

MMR and hereditary non-polyposis colorectal cancer (HNPCC)
Defects in MMR are implicated in hereditary non-polyposis colorectal cancer (HNPCC), also termed Lynch syndrome, and less frequently in endometrial, ovarian, gastric and some other cancer forms.Colon epithelium has the highest known proliferation rate of all cell types, and this may directly contribute to the accumulation of MMR-deficiency-induced replication errors specifically in this tissue type.Lynch syndrome accounts for about 5-8% of all colon cancer cases.About 500 different Lynch syndrome-associated MMR gene mutations have been found, and among them MLH1, MSH2, and MSH6 gene mutations constitute about 50%, 40%, and 10%, respectively (for mutations found in respective genes see http: //www.insight-group.org/).Mutations in MSH6 show stronger association with endometrial than colon cancer (Wijnen et al., 1999).Mice defective in either MLH1, MSH2 or MSH6 show cancer susceptibility and develop mainly lymphomas, gastrointestinal (GI) epithelial adenomas or basal cell carcinomas, whereas PMS2 −/− mice develop lymphomas and sarcomas, but not GI tumors.Moreover, a few pathogenic germline muta-tions have been found in PMS2 and most of them are associated with Turcot syndrome, characterized by brain tumors, colonic polyps and colon cancer.Finally, mutations in MLH3 may also be associated with Lynch syndrome.On the other hand, although MSH3 frequently shows somatic mutations in MSIpositive tumors, and potentiates the consequences of defects in other MMR genes, MSH3-deficient mice are not cancer prone and no Lynch syndrome-associated germline mutations have been found in MSH3 (Lynch & de la Chapelle, 1999;Peltomaki, 2005;Chao & Lipkin, 2006).

CooPeRATIoN beTweeN hMTH1 PRoTeIN AND MMR
The antimutagenic role of MTH1 is less pronounced than that of the MutT protein, since while the mutT E. coli mutant has a specifically increased level of spontaneous mutations by 100-to 10 000-fold, their level in the Hprt locus of homozygous MTH1 −/− mouse cells is increased only 2 times in comparison with the wild type MTH1 +/+ mouse cells (Tsuzuki et al., 2001a;2001b).This low level of spontaneous mutations can be explained, at least partially, by the existence of MutT homologue 2 (MTH2) protein, which is active on 8-oxodGTP and may backup MTH1 function (Cai et al., 2003), and by the existence of NUDT5 protein.Importantly, both proteins, i.e. mouse MTH2 and human NUDT5 expressed in the mutT E. coli strain suppressed its mutator phenotype (Cai et al., 2003;Ishibashi et al., 2003).Furthermore, mutagenesis may be prevented by efficient removal of oxidized bases incorporated from the cellular dNTP pool by DNA repair systems, such as BER, MMR or NER.Interestingly, although the MTH1 −/− mice showed a higher incidence of lung, liver and stomach cancer (Tsuzuki et al., 2001a;2001b), they did not show an increased frequency of spontaneous rpsL − forward mutations in comparison with the wild type MTH1 +/+ mice.On the other hand, the frequency of AT→CG transversions was 3.6-times higher, and of singlebase frameshifts at mononucleotide runs 5.7-times higher in MTH1 −/− than in MTH1 +/+ mice (Egashira et al., 2002).Single-base frameshifts mononucleotide runs are a characteristic feature of MMR deficiency, and MMR was in fact shown to remove 8-oxo-dGMP incorporated from the nucleotide pool, since overexpression of hMTH1 reduced the DNA 8-oxoG level in MSH2 −/− cells (i.e.MMR-defective) (Colussi et al., 2002;Russo et al., 2004).Furthermore, MTH1 −/− MSH2 −/− mice in comparison with MSH2 −/− once had a specifically increased occurrence of GC→TA transversions, which could be induced by 2-oxodAMP incorporation opposite G, or by erroneous incorporation of dAMP opposite 8-oxoG present in DNA (Egashira et al., 2002).Moreover, hMTH1 overexpression reduced the level of spontaneous Hprt locus mutations in the MSH2 −/− background, and among them the highest reduction was observed in the case of frameshifts, AT→GC transitions and AT→ TA and GC→TA transversions (Russo et al., 2004).The AT→TA and GC→TA transversions are induced by erroneous incorporation of dAMP and dCMP, respectively, opposite template 2-oxoA (Kamiya & Kasai, 1997a;1997b;Barone et al., 2007).Consistently, MMR could be involved in 2-oxoA removal, since MutSα has been shown to bind 2-oxoA-containing DNA (Barone et al., 2007).Therefore, the low level of spontaneous mutations in MTH1 −/− mice and mouse cultured cells can be attributed partially to the removal, by the MMR system, of oxidized bases incorporated from the nucleotide pool.Furthermore, in the MTH1 −/− background the high level of oxidized bases incorporated to DNA from the nucleotide pool may partially sequester MMR, leading to a more frequent occurrence of single-base frameshifts at mononucleotide runs (Egashira et al., 2002).Surprisingly, AT→CG transversions, which are dramatically increased in mutT E. coli cells, were only slightly increased in MTH1 −/− mice (Egashira et al., 2002), and only slightly decreased by hMTH1 overexpression in MSH2 −/− mice (Russo et al., 2004).AT→CG transversions are induced by 8-oxodGMP incorporation opposite template A, and in mutT E. coli cells, lacking the MutT protein which removes both 8-oxodGTP and 8-oxodGDP, they can be much more frequent than in MTH1 −/− cells, lacking the 8-oxodGTP-hydrolysing MTH1 protein, but still possessing the MTH2 and NUDT5 proteins dephosphorylating 8-oxodGTP and 8-oxodGDP, respectively.On the other hand, MTH1 −/− mice showed increased levels of all types of mutations connected with 2-oxoA, consistently with the fact that human MTH1 hydrolyses 2-oxodATP (Russo et al., 2004).This may suggest that MTH1 is the only protein specifically involved in 2-oxodATP elimination from the mammalian cellular nucleotide pool or that 2-oxoA repair by MYH glycosylase (Ohtsubo et al., 2000) or MMR is saturated upon MTH1 deficiency.
figure 1. Prevention of transversion mutations by mammalian Go system (adapted from scheme proposed for E. coli byMichaels and Miller, 1992).8-OxoG is formed in DNA both by direct guanine oxidation and by 8-oxodGTP incorporation from nucleotide pool.8-OxodGTP is incorporated mainly opposite A, and thus, if unrepaired may lead to AT→CG transversion.When 8-oxoG is present in DNA it may pair with A upon replication, which leads to GC→TA transversions.hMTH1 pyrophosphohydrolase, and hOGG1, hOGG2 and hMYH glycosylases act together to prevent these mutations.E. coli cells lack hOGG2 protein, 8-oxodGTP is hydrolyzed by MutT protein, 8-oxoG is excised from pair with C by MutM glycosylase, and A is excised from pair with 8-oxoG by MutY glycosylase.G*, 8-oxoG.

figure 3 .
figure 3. Models of MMR complex assembly (A, b and C) and mismatch excision (D).(A)In the molecular-switch model(Gradia et al., 1999) MutSα normally exists in ADP-bound form, and upon mismatch binding ADP is exchanged to ATP, which induces conformational changes and ATP-independent diffusion of multiple MutSα (possibly in complex with MutLα) sliding clamps along DNA helix.(B) In the active-translocation model(Blackwell et al., 1998;Martik et al., 2004), MutSα clamp (possibly in complex with MutLα) translocates along DNA and ATP is used as energy source to drive its motion.(C) In the DNA bending/verification model(Wang & Hays, 2003; 2004)   MutSα remains at the mismatch and makes contact with strand break through DNA bending.MutS is able to sense the mismatch, and when bound to DNA without mismatch it hydrolyzes ATP and in consequence becomes displaced from the DNA.On the other hand, when MutS binds DNA containing mismatch, ATP is not hydrolyzed, what leads to activation of downstream MMR effectors.(D) When MutSα-MutLα complex comes into contact with PCNA and RFC bound to strand break, it triggers further MMR steps.MutSα, PCNA and RFC activate latent MutLα endonuclease(Kadyrov et al., 2006), which cleaves DNA at both sides of the mismatch in ATP-dependent manner.Next, at the 5'-break (created by MutLα endonucleolytic cleavage or preexisting in DNA) MutSα-MutLα complex displaces RFC from complex with PCNA and loads Exo1, which hydrolyzes DNA in 5'→3' direction.The single stranded gap is stabilized by RPA.RPA reduces Exo1 processivity by binding ssDNA, and Exo1 is further inhibited by MutSα and MutLα upon reaching the mismatch.This leads to termination of excision.Finally, gap is filled by Pol δ, in presence of PCNA and RPA, and ends are joined by LIG1.

Table 1 . overview of bacterial MutT and MMR proteins and their eukaryotic counterparts
*S. cerevisiae cells are lack of homologue of E. coli DNA Pol III; ** human cells are lack of homologue of E. coli DNA Pol III.The main DNA polymerase, Polδ carries on the repair synthesis.
The single stranded gap is stabilized by RPA.RPA reduces Exo1 processivity by binding ssDNA, and Exo1 is further inhibited by MutSα and MutLα upon reaching the mismatch.This leads to termination of excision.Finally, gap is filled by Pol δ, in presence of PCNA and RPA, and ends are joined by LIG1.