||Ohno Mizuki, Sakumi Kunihiko, Ryutaro Fukumura, Masato Furuichi, Yuki Iwasaki, Masaaki Hokama, Toshimichi Ikemura, Teruhisa Tsuzuki, Yoichi Gondo, Yusaku Nakabeppu, 8-Oxoguanine causes spontaneous de novo germline mutations in mice, Scientific reports, 10.1038/srep04689, 4, 2014.04, Spontaneous germline mutations generate genetic diversity in populations of sexually reproductive organisms, and are thus regarded as a driving force of evolution. However, the cause and mechanism remain unclear. 8-oxoguanine (8-oxoG) is a candidate molecule that causes germline mutations, because it makes DNA more prone to mutation and is constantly generated by reactive oxygen species in vivo. We show here that endogenous 8-oxoG caused de novo spontaneous and heritable G to T mutations in mice, which occurred at different stages in the germ cell lineage and were distributed throughout the chromosomes. Using exome analyses covering 40.9 Mb of mouse transcribed regions, we found increased frequencies of G to T mutations at a rate of 2 × 10-7 mutations/base/generation in offspring of Mth1/Ogg1/Mutyh triple knockout (TOY-KO) mice, which accumulate 8-oxoG in the nuclear DNA of gonadal cells. The roles of MTH1, OGG1, and MUTYH are specific for the prevention of 8-oxoG-induced mutation, and 99% of the mutations observed in TOY-KO mice were G to T transversions caused by 8-oxoG; therefore, we concluded that 8-oxoG is a causative molecule for spontaneous and inheritable mutations of the germ lineage cells..
||Behmanesh M, Sakumi K, Abolhassani N, Toyokuni S, Oka S, Ohnishi YN, Tsuchimoto D, Nakabeppu Y., ITPase-deficient mice show growth retardation and die before weaning, Cell Death and Differentiation, 16(10):1315-1322., 2009.10.
||Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M, Nakabeppu Y, Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption., Cancer Res., 63, 5, 902-905, 63(5):902-905, 2003.01.
||Akiko Shiraishi, Sakumi Kunihiko, Mutsuo Sekiguchi, Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase, Carcinogenesis, 21, 10, 1879-1883, 2000.10, O6-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the Mgmt gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. Mgmt(-/-) mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD50 values of Mgmt(-/-) and Mgmt(+/+) mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of Mgmt(-/-) mice to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of Mgmt(+/+) and Mgmt(-/-) mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O6-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyl-transferase. transferase. Mgmt(-/-) mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are Mgmt(+/+) mice. Thus, the level of O6-methylguanine-DNA methyl-transferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation..
||Sakumi Kunihiko, Akiko Shiraishi, Seiichiro Shimizu, Teruhisa Tsuzuki, Takatoshi Ishikawa, Mutsuo Sekiguchi, Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice, Cancer Research, 57, 12, 2415-2418, 1997.06, Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase Tsuzuki et al., Carcinogenesis (Lond), 17: 1215-1220, 1996]. These MGMT(-/-) mice were most sensitive to the alkylating carcinogen, methylnitrosourea; when varied doses of methylnitrosourea were administered to 6-week-old mice and survivals at the 30th day were determined, LD50s of MGMT(-/-) and MGMT(+/+) mice were 20 and 240 mg/kg of body weight, respectively. MGMT(+/-) mice were as resistant as MGMT(+/+) mice, but some difference in survival time was noted when the two genotypes of mice were exposed to a relatively high dose of methylnitrosourea. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT(-/-) mice exposed to methylnitrosourea at a dose of 2.5 mg/kg of body weight. In case of exposure to the same dose of drug, no or few tumors occurred in the MGMT(+/+) and MGMT(+/-) mice. It appears that the DNA repair methyltransferase protein protected these mice from methylnitrosourea-induced tumorigenesis..
||Sakumi Kunihiko, Masato Furuichi, T. Tsuzuki, T. Kakuma, Shun-Ichiro Kawabata, H. Maki, M. Sekiguchi, Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo- dGTP, a mutagenic substrate for DNA synthesis, Journal of Biological Chemistry, 268, 31, 23524-23530, 1993.07, 8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues. Human cells contain enzyme activity, which hydrolyzes 8- oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA. We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence. We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence. The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity. When the human cDNA was expressed in E. coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity. In such cells, the frequency of spontaneous mutation was greatly reduced. We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals..
||Sakumi Kunihiko, Mutsuo Sekiguchi, Regulation of expression of the ada gene controlling the adaptive response. Interactions with the ada promoter of the Ada protein and RNA polymerase, Journal of Molecular Biology, 10.1016/0022-2836(89)90348-3, 205, 2, 373-385, 1989.01, The Ada protein of Escherichia coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada. Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene. To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RNA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment carrying the ada promoter region. Footprinting analyses revealed that the methylated Ada protein binds to a region from positions -63 to -31, which includes the ada regulatory sequence AAAGCGCA. No firm binding was observed with the non-methylated Ada protein, although some DNase I-hypersensitive sites were produced in the promoter by both types of Ada protein. RNA polymerase did bind to the promoter once the methylated Ada protein had bound to the upstream sequence. To correlate these phenomena with the process in vivo, we used the DNAs derived from promoter-defective mutants. No binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position -47 within the ada regulatory sequence. In the case of a -35 box mutant with a T to A change at position -34, the methylated Ada protein did bind to the ada regulatory sequence, yet there was no RNA polymerase binding. Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promoter. The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321. The role of methylation of each cysteine residue was investigated using mutant forms of the Ada protein. The Ada protein with the cysteine residue at position 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated. When the Ada protein with alanine at position 321 was methylated, it acquired the potential to bind to the ada promoter. These results are compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby facilitating binding of the protein to the upstream regulatory sequence..
||Sakumi Kunihiko, Yusaku Nakabeppu, Y. Yamamoto, Shun-Ichiro Kawabata, S. Iwanaga, M. Sekiguchi, Purification and structure of 3-methyladenine-DNA glycosylase I of Escherichia coli, Journal of Biological Chemistry, 261, 33, 15761-15766, 1986.12, We constructed a recombinant plasmid carrying a gene that suppresses tag mutation. To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8. 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-β-D-thiogalactopyranoside. From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined. The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method. It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. Only 3-methyladenine was excised from methylated DNA by the purified glycosylase. These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I..