Mouse methyltransferase for repair of O6-methylguanine and O4-methylthymine in DNA.
cDNA for mouse O6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8 x 10(4) molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost equal efficiency. In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppressed by the function of mouse methyltransferase, in vivo.
Kawate
H
H
Department of Biochemistry, Kyushu University, Fukuoka, Japan.
Ihara
K
K
Kohda
K
K
Sakumi
K
K
Sekiguchi
M
M
D016475:3T3 Cells / Q000201:enzymology / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007424:Intracellular Fluid / Q000201:enzymology; D008769:Methylnitronitrosoguanidine; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
D018076:DNA, Complementary; D008769:Methylnitronitrosoguanidine; C021223:O-4-methylthymine; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine