piperidines and dimethyl-sulfate

Topics: Binding Sites; DNA; DNA Damage; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Guanine; Piperidines; Protein Binding; Sequence Analysis, DNA; Sulfuric Acid Esters; Taq Polymerase

Topics: Base Sequence; Chemical Phenomena; Chemistry; Cloning, Molecular; DNA; Gene Expression Regulation; Genes; Hydrazines; Mutation; Piperidines; Recombination, Genetic; Repressor Proteins; RNA Splicing; RNA, Messenger; Sulfuric Acid Esters

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane 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 pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.

Topics: Alkylating Agents; Alkylation; Base Sequence; Cell Line, Transformed; Chromatin; Dimethyl Sulfoxide; DNA; DNA Damage; DNA Footprinting; DNA Methylation; Exons; Fragile X Mental Retardation Protein; Guanine; Humans; Lymphocytes; Molecular Conformation; Molecular Sequence Data; Nerve Tissue Proteins; Piperidines; Potassium Permanganate; Promoter Regions, Genetic; Purines; RNA-Binding Proteins; Sulfones; Sulfuric Acid Esters

The repressor proteins BlaI and MecI bind similarly to the bla operator implicated in the regulation of beta-lactamase synthesis in Staphylococcus aureus. BlaI binds to two separate dyads but neither copper-phenanthroline footprinting nor dimethyl sulphate (DMS) methylation protection assays produced any evidence of a change in the geometry of the DNA between the two dyads. It is concluded that BlaI molecules bound at the dyads probably do not cause bending or looping of the intervening DNA. DMS protection assays of BlaI binding to the bla operator in vitro and in vivo gave similar results so that it is tentatively concluded that the in vitro results are an accurate reflection of the in vivo situation. Deletion of the dyad nearest to the blaZ gene resulted in decreased synthesis of the chloramphenicol acetyltransferase reporter protein synthesized from the blaZ promoter/translation initiator. Explanations for this are considered.

Topics: Bacterial Proteins; Base Sequence; beta-Lactamases; Chloramphenicol O-Acetyltransferase; Deoxyribonuclease I; DNA; DNA Footprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Bacterial; Genes, Reporter; Molecular Sequence Data; Nucleic Acid Conformation; Operator Regions, Genetic; Phenanthrolines; Piperidines; Plasmids; Promoter Regions, Genetic; Repressor Proteins; Sequence Deletion; Staphylococcus aureus; Sulfuric Acid Esters; Time Factors

Topics: Alkylating Agents; Animals; Base Sequence; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Glycosylases; Embryo, Mammalian; Fibroblasts; Genetic Techniques; Mice; Molecular Sequence Data; N-Glycosyl Hydrolases; Piperidines; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Binding; RNA, Long Noncoding; RNA, Untranslated; Sulfuric Acid Esters; Ultraviolet Rays

Nitrogen mustards such as mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures.

Topics: Alkylating Agents; Alkylation; Base Pair Mismatch; Bisbenzimidazole; Cross-Linking Reagents; DNA; Guanine; Hydrogen-Ion Concentration; Mechlorethamine; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Piperidines; Sulfuric Acid Esters

In the presence of alkali cations, notably potassium and sodium, DNA oligomers that possess two G-rich repeats associate into either a tetrameric parallel G-quadruplex or a variety of dimeric antiparallel G-quadruplexes. The formation of such structures is normally a very slow process. Some proteins, such as the beta-subunit of the Oxytricha telomere-binding protein, promote the formation of G-quadruplex structures in a chaperone-like manner. In this report, we present data concerning the role of a perylene derivative, PIPER, in the assembly of G-quadruplex structures as the first example of a small ligand behaving as a driver in the assembly of polynucleotide secondary structures. Gel-shift experiments demonstrate that PIPER can dramatically accelerate the association of a DNA oligomer containing two tandem repeats of the human telomeric sequence (TTAGGG) into di- and tetrameric G-quadruplexes. In so doing, PIPER alters the oligomer dimerization kinetics from second to first order. The presence of 10 microM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold from 2 microM oligomer. These results imply that some biological effects elicited by G-quadruplex-interactive agents, such as the induction of anaphase bridges, may stem from the propensity such compounds have for assembling G-quadruplexes.

Topics: Anthracenes; Cations, Monovalent; Dimerization; DNA; DNA Footprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; G-Quadruplexes; Guanine; Humans; Macromolecular Substances; Nucleic Acid Conformation; Perylene; Piperidines; Potassium; Sulfuric Acid Esters; Tandem Repeat Sequences; Telomere

Topics: Animals; Base Sequence; Cattle; Cloning, Molecular; DNA Footprinting; DNA Probes; DNA-Binding Proteins; DNA, Mitochondrial; Genome; Indicators and Reagents; Methylation; Mitochondria; Molecular Sequence Data; Piperidines; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Sulfuric Acid Esters

Topics: Acetates; Acetic Acid; Cacodylic Acid; Chemical Precipitation; Electrophoresis, Polyacrylamide Gel; Hydrazines; Piperidines; Sequence Analysis, DNA; Sulfuric Acid Esters

We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer.

Topics: Alkylation; DNA; DNA Damage; Escherichia coli; Gene Amplification; Guanine; Lac Operon; Molecular Sequence Data; Oligonucleotides; Piperidines; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Sulfuric Acid Esters

Topics: Diet; Parasympatholytics; Peptic Ulcer; Piperidines; Sulfuric Acid Esters

Topics: Benzhydryl Compounds; Parasympatholytics; Piperidines; Sulfuric Acid Esters