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Additional file 4: Figure S4. Positions of mutations in the gpt gene in the small intestine of Msh2-bearing mice (M(+/+)) carrying gpt and Msh2-KO mice (M(−/−)) carrying gpt. Potassium bromate (1.5 g/L) in the drinking water or the vehicle control (Control) was administered for 28 days. Numbers in the column next to the doses are animal identification numbers shown in Additional Table S1. Letters A, G, T, and C indicate base substitutions, and the number of letters in brackets are the number of clonal mutants. Arrows indicate the positions of insertion of base(s). d and (AA) indicates deletion of base and insertion of two adenine residues, respectively. Red letters in the gpt sequence indicate nucleotides whose mutations were induced.

Additional file 5: Table S1. Mutant frequencies in the small intestine of Msh2-bearing or -KO mice carrying gpt to which vehicle or potassium bromate was administered via the drinking water.

Additional file 3: Figure S3. Screenshot of the 17qB2 region of an Msh2-KO mouse (animal #1 in the vehicle control group; see also Additional Table S1) as shown in Integrative Genomics Viewer. All sequence reads were mapped against a reference sequence (mm10 + lambda EG10). Only chimeric reads (Lambda EG10: genomic or genomic: Lambda EG10) are shown in this figure. Junction 1 and 2 indicate presumed integration sites of the gpt gene. A junction is a site boundary between the mouse genome sequence and the Lambda EG10 sequence within a chimeric read. The two junctions mapped on the mm10 reference sequence were consistent with two previously reported junctions reported using the mm9 reference sequence [13].

Additional file 1: Figure S1. Representative Msh2 genotyping results. The genotype of each mouse was determined by polymerase chain reaction (PCR) using (a) the primer 1–4 and (b) the primer 5–7 described in Materials and Methods. WT and KO indicate the PCR product of the Msh2 wild-type allele and Msh2 KO allele, respectively, and numbers indicated in parenthesis are the size (bp, base pair) of amplicons. ‘+/+’, ‘+/−’, and ‘−/−’ indicate the Msh2 genotype of each animal, as determined by PCR. Dose, A dose of potassium bromate (g/L) to which each mouse was administered. ID, ID of the animal in the treatment groups, as shown in Additional Table S1.

Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is further enhanced by ionizing radiation and by various chemicals. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is the most abundant, and appears to play important roles in mutagenesis and carcinogenesis. We have focused on following the two enzymes. MTH1 protein hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. On the other hand, MUTYH protein, having adenine/2-hydroxyadenine DNA glycosylase activity, is expected to prevent G:C to T:A transversions, by excising adenine from G:A mismatches induced by 8-oxoG and 2-OH-A. To analyze the function of the mammalian MTH1 and MUTYH proteins in vivo, we established gene-knockout mice for these two enzymes, and investigated spontaneous tumorigenesis as well as mutagenesis.

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.