Evidence that covalent complex formation between BCNU-treated oligonucleotides and E. coli alkyltransferases requires the O6-alkylguanine function.
Chloroethylnitrosoureas (CENUs) are thought to induce cytotoxic DNA interstrand cross-links via an initial reaction at O6-position of guanine, yielding a rearranged intermediate, O6,N1-ethanoguanine. Repair of these adducts by mammalian and bacterial DNA alkyltransferases blocks the formation of cross-links. Human alkyltransferase can form a covalent complex with DNA containing BCNU-induced cross-link precursors, but the nature of the DNA-protein linkage remains unknown. Using E. coli alkyltransferases expressed by the ada and ogt genes, we now demonstrate that both enzymes can form such complexes with CENU-treated DNA. We attribute this reaction to the O6-alkylguanine repair function, because an N-terminal fragment of the ada protein, which has only alkylphosphotriester repair activity, failed to form a similar complex. This result is consistent with the idea that complex formation requires an alkyltransferase reaction with a guanine adduct, such as O6,N1-ethanoguanine. It tends to exclude the possibility that such reactions simply involve alkylation of the enzyme by reactive DNA adducts such as chloroethylphosphate or chloroethylguanine.
Gonzaga
P E
PE
Department of Biochemical and Clinical Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38101.
Harris
L
L
Margison
G P
GP
Brent
T P
TP
D001483:Base Sequence; D002330:Carmustine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine