Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultu red human cells (in vivo) and in vitro to probe DNA-protein interactions al ong sequences of the promoter and exon 1 of the Fragile-X mental retardatio n 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treat ing cultured cells tin vivo) and naked DNA (in vitro) with S(N)1 (N-methyl- N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-n itrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), metha ne sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfo nic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites whe re DNA-protein interactions are observed. In these regions, the S(N)1 methy lating agent reactions produced only hypo-reactive sites. In contrast, iodo alkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper -reactive sites. Although there are hyper-reactive sites for the other S(N) 2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounc ed for the sulfate and sulfonate-derived alkylating agents than for the iod oalkanes. However, DMS modification in the presence of methyl sulfone, a co mpound that does not alkylate DNA, eliminates the hyper-reactive site obser ved at +14. This suggests that the electron-rich oxygen atoms of the sulfat e and sulfonate-derived S(N)2 alkylating agent structure position the alkyl ating moiety to the neighboring N-7-guanine position to favor alkyl transfe r to the guanine. Using KMnO4 to probe for single-strand DNA, an unpaired c ytosine base was detected at the 5'-side of the hyperreactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. Ln conclusion, we show that the sequence context-dependent formatio n of alkylpurines is determined by the chemical nature of the alkylating ag ent, the DNA sequence context, chromatin structure, and the presence of oth er non-reactive molecules that can inhibit alkylation. (C) 2001 Academic Pr ess.