Mutsuo Sekiguchi

Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli

8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is a potent mutagenic substrate for DNA synthesis. The accumulation of 8-oxo-dGTP in the nucleotide pool induces G:C-->T:A transversion as well as A:T-->C:G transversion, and Escherichia coli cells possess mechanisms for preventing such mutations. The mutT gene product specifically hydrolyzes 8-oxo-dGTP to the monophosphate form while the mutM and the mutY gene products function to correct mispairs caused by incorporation of 8-oxoguanine into DNA. From analyses of forward mutations induced in cells lacking 8-oxo-dGTPase (MutT protein) and/or repair enzymes that suppress mutations caused by 8-oxoguanine in DNA (MutM and MutY proteins), cooperative functions of these proteins in control of the spontaneous mutagenesis became evident. In mutator strains lacking MutT and/or MutM proteins, 8-oxoguanine of DNA increased to a concentration expected from the increased rate of mutation.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

Three-Dimensional Structure of a DNA Repair Enzyme, 3-Methyladenine DNA Glycosylase II, from Escherichia coli

The three-dimensional structure of Escherichia coli 3-methyladenine DNA glycosylase II, which removes numerous alkylated bases from DNA, was solved at 2.3 A resolution. The enzyme consists of three domains: one alpha + beta fold domain with a similarity to one-half of the eukaryotic TATA box-binding protein, and two all alpha-helical domains similar to those of Escherichia coli endonuclease III with combined N-glycosylase/abasic lyase activity. Mutagenesis and model-building studies suggest that the active site is located in a cleft between the two helical domains and that the enzyme flips the target base out of the DNA duplex into the active-site cleft. The structure of the active site implies broad substrate specificity and simple N-glycosylase activity.

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MutT‐related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8‐oxo‐ 7,8‐dihydrodeoxyguanosine triphosphate (8‐oxo‐dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8‐oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8‐oxo‐dGDP to monophosphate with a Km of 0.77 µM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8‐oxo‐dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT− cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8‐oxoguanine in human cells.

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with Vmax/Km values of 1.3 × 10−3 and 1.7 × 10−3, respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1–0.5 × 10−3). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.

Molecular cloning of AtMMH, an Arabidopsis thaliana ortholog of the Escherichia coli mutM gene, and analysis of functional domains of its product

Abstract We isolated and characterized cDNAs and a genomic clone encoding an Arabidopsis thaliana MutM homolog (AtMMH). AtMMH is a single-copy gene spanning about 3 kb in the nuclear genome, and comprises ten exons. The AtMMH gene encodes two types of mRNA (AtMMH-1 and AtMMH-2) formed by alternative splicing of exon 8. Western analysis of a crude extract from leaves of A. thaliana, using polyclonal antibodies against the recombinant proteins, demonstrated the presence in vivo of a single 44-kDa polypeptide that comigrates with the product of in vitro translation of the AtMMH-1 mRNA. AtMMH-1 protein prepared in vitro is able to nick double- stranded oligonucleotides containing 8-oxo-7,8-dihydroguanine (8-oxoG) and to bind such oligonucleotides, as does the Escherichia coli MutM protein, which possesses 8-oxoG DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities. Deletion of six amino acids (PELPEV), which are conserved among all known MutM homologs, from the N-terminal end of the AtMMH-1 protein abolishes its nicking but not its DNA-binding activity, indicating that these residues are essential for catalytic activity. Although the AtMMH-1 protein has a unique structure at its C-terminal end, which consists of alternating repeats of basic and acidic amino acids, this structure is dispensable for activity. However, the adjacent amino acid sequence (residues 268 to 281) is essential for repair activity.

Regulation of Expression of the Human MTH1 Gene Encoding 8-Oxo-dGTPase ALTERNATIVE SPLICING OF TRANSCRIPTION PRODUCTS

The enzyme 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxo-dGTP into DNA. We investigated expression of MTH1gene encoding 8-oxo-dGTPase. Large amounts ofMTH1 mRNA were present in thymus and testis, embryonic tissues, and certain cell lines. In peripheral blood lymphocytes, the level of MTH1 mRNA was significantly increased after concomitant treatment with phytohemagglutinin and interleukin-2. Analyses of the 5′ regions of the MTH1 transcripts revealed that 7 types of MTH1 mRNAs, which may be produced by transcription initiation at different sites and/or alternative splicing. The MTH1 gene consists of 5 major exons, some of which are composed of differentially processed segments. All types ofMTH1 mRNAs carry the entire coding region, and may be functional. Three ATG initiation codons in-frame were found in the 5′ regions of some of the MTH1 mRNAs. There is a polymorphic alteration at the 5′ splicing site (GT to GC) located in exon 2, an event which affects splicing patterns of the MTH1transcript. Allele frequency of this polymorphism is about 20% among healthy volunteers.

Deficient expression ofO 6-Methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and hMSH2 is related to poor prognosis in human biliary tract carcinoma

BackgroundO6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups fromO6-methylguanine to itself. Alkylation of DNA at theO6 position of guanine is an important step in the induction of mutations in the organism by alkylating agents. TheO6-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location.MethodsWe examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining.ResultsMGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types.ConclusionsCombined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.

Human MTH3 (NUDT18) Protein Hydrolyzes Oxidized Forms of Guanosine and Deoxyguanosine Diphosphates: COMPARISON WITH MTH1 AND MTH2*

Background: An oxidized guanine, 8-oxo-7,8-dihydroguanine, induces base mispairing, thereby altering genetic information. Results: Human MTH3 degrades 8-oxoguanine-containing nucleoside diphosphates to prevent misincorporation of 8-oxoguanine into DNA and RNA. Conclusion: MTH3 is closely related to MTH1 and MTH2 but differs in substrate specificity. Significance: MTH3 may be involved in maintaining the high fidelity of DNA replication as well as transcription under oxidative stress. Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.