A unique structural feature of rabbit DNA repair methyltransferase as revealed by cDNA cloning.
cDNA encoding rabbit O6-methylguanine-DNA methyltransferase that repairs DNA damaged by alkylating agents was isolated, using as a probe a fragment of mouse cDNA coding for a region containing the active site of the enzyme. The nucleotide sequence of the cDNA revealed that the rabbit methyltransferase is a 181-amino acid polypeptide with a mol. wt of 19,385. Expression of the cDNA in a methyltransferase-deficient Escherichia coli mutant resulted in appearance of a 23 kDa polypeptide with methyltransferase activity, and this rendered the E.coli cells resistant to N-methyl-N'-nitro-N-nitrosoguanidine, in terms of both cell killing and induction of mutation. The rabbit methyltransferase is highly homologous to this enzyme in human, mouse, rat and hamster, but is 26-30 amino acid residues shorter as compared with methyltransferases in other mammalian species. Based on a comparison of the nucleotide sequences for the C-terminal regions of these proteins, we propose that a single base substitution, which would generate a TGA termination codon, was introduced into the sequence for the rabbit enzyme during the process of evolution. Existence of the naturally occurring truncated form of methyltransferase suggests that the longer C-terminal tails of other mammalian methyltransferases may have no significant role in exerting functions of the enzyme in vivo.
Iyama
A
A
Department of Biochemistry, Kyushu University, Fukuoka, Japan.
Sakumi
K
K
Nakabeppu
Y
Y
Sekiguchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase