Sequence-selective depurination, DNA interstrand cross-linking and DNA strand break formation associated with alkylated DNA.
Alkylation of DNA by chloroethylnitrosourea (CNU) at the guanine N7 position has been shown to occur in a sequence-selective fashion. In this report we find that the depurination of these alkylated sites occurs with two distinct kinetic components--GG sequences depurinate within 30 min of exposure to CNU, while depurination at GT sequences is first observed after 1 h and continues to increase 16 h after drug exposure. These apurinic sites are converted to DNA strand breaks and constitute less than 10% of the total sites of guanine N7 alkylation. Spermidine was found to decrease alkylation in 5'-GG-3' sequences but increases alkylation at 5'-GTC-3' sequences. These findings suggest that the majority of the guanine N7 alkylations formed by CNU are stable, with a minor adduct being responsible for the slow depurination event. We propose that the rapid depurination induced by CNU occurs from an initial guanine O6 alkylation, which then depurinates via a guanine O6-N7 cyclized intermediate. We also propose that the resulting apurinic sites may lead to DNA interstrand cross-linking (ISC). In support of these hypotheses we show that (i) DNA modified with the monoalkylating agent dimethylsulfate forms DNA ISC upon depurination; (ii) ellagic acid enhances the level of guanine N7 alkylation and alters the pattern of sequence selectivity shown by three bifunctional chloroethylating agents CNU, mitozolomide and methyl 3-(2-chloroethyl)-4-oxoimidazo[5,1-d]-1,2,3,5-tetrazine-8-ca rboxylate but not with nitrogen mustard; (iii) ellagic acid has no effect upon the frequency of alkylation observed with the monofunctional alkylators N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and methylmethanesulfonate; (iv) ellagic acid increases the frequency of depurination and strand break formation induced by CNU without affecting the sequence-selective pattern of depurination.
Prakash
A S
AS
School of Pharmacy, University of Southern California, Los Angeles 90033.
Gibson
N W
NW
D000478:Alkylation; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004610:Ellagic Acid / Q000494:pharmacology*; D005038:Ethylnitrosourea / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000378:metabolism; D008969:Molecular Sequence Data; D013095:Spermidine / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004610:Ellagic Acid; D006147:Guanine; D004247:DNA; D008741:Methyl Methanesulfonate; D005038:Ethylnitrosourea; D013095:Spermidine