Interaction of DNA methyltransferase with aminofluorene and N-acetylaminofluorene modified poly(dC-dG).
Poly(dC-dG) was reacted in vitro to yield templates containing similar amounts of aminofluorene (AF) or acetylaminofluorene (AAF) adducts. These modified poly(dC-dG) templates were tested in an in vitro DNA methylation system utilizing 1500-fold purified rat liver methyltransferase (DMase) to compare and quantitate the effects of these adducts on the kinetics of methylation and the interaction of DMase with such templates. Enzymatic methylation is severely impaired by arylamine adducts, with bound AF inhibiting more than AAF (relative Vmax 0.24 for AAF-poly(dC-dG) and 0.066 for AF-poly(dC-dG). The apparent km for the reaction is not appreciably altered by AAF modification: 10 microM for dCdG dinucleotide units, but it is threefold lower (3 microM) for AF-poly(dC-dG). In competition experiments it was demonstrated that a translocational block is imposed by the adducts. From differential salt inhibition assays and preincubation assays, no change in the ionic binding to the altered templates could be detected, which suggests that the enzyme interacts very strongly through hydrophobic interactions with the fluorene ring. Evidence that the fluorene ring is exposed is supported by circular dichroism spectra of the templates under the conditions of the assay, which indicated that the AF adducts do not appreciably change the normal B conformation of the template, while the template with 9.5% modification by AAF adducts adopted a Z form. These results suggest that the inhibitory effects of AAF and, in particular, AF upon DMase-catalyzed methylation reactions are not dependent upon helix conformation. Instead, they appear to depend upon DMase recognition of an altered dG base configuration, which is responsible for altered binding and methylation kinetics.
D015073:2-Acetylaminofluorene / Q000494:pharmacology*; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D002942:Circular Dichroism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D005449:Fluorenes / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism; D013698:Templates, Genetic
D005449:Fluorenes; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); C012177:2-aminofluorene; D012436:S-Adenosylmethionine; D015073:2-Acetylaminofluorene; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases