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.
Department of Biochemical and Clinical Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38101.
P E
PE
Gonzaga
L
L
Harris
G P
GP
Margison
T P
TP
Brent
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