Mutator phenotype of MUTYH-null mouse embryonic stem cells.
To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.
Hirano
Seiki
S
Division of Neurofunctional Genomics, Medical Institute of Bioregulation, Kyushu University, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Fukuoka, 812-8582, Japan.
Tominaga
Yohei
Y
Ichinoe
Akimasa
A
Ushijima
Yasuhiro
Y
Tsuchimoto
Daisuke
D
Honda-Ohnishi
Yoko
Y
Ohtsubo
Toshio
T
Sakumi
Kunihiko
K
Nakabeppu
Yusaku
Y
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000166:cytology*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D018095:Germ-Line Mutation; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
D018076:DNA, Complementary; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C437225:adenine glycosylase; C061738:mutY adenine glycosylase