Action de la moutarde azotée sur le DNA utilisé comme matrice pour la synthèse du RNA

Abstract

The goal of this study is to elucidate the mechanism by which nitrogen mustard, a difunctional alkylating agent, inhibits DNA template activity for RNA synthesis. This inhibition could be due not only monofunctional alkylations, known to hinder RNA synthesis, but also to interand intrastrand bridges formed by difunctional alkylation. DNA from bacteriophage T, and Escherichia coli RNA polymerase provide a well-characterized system for RNA synthesis in vitro. Only 20°/, of the T, genome is transcribed by E . coli RNA polymerase and hence only those alkylations falling in this region could influence RNA synthesis.Three types of experiments were carried out. T, DNA was alkylated by treatment with varying concentrations of nitrogen mustard. This alkylated DNA was used as template for RNA synthesis; the extent of interstrand crosslinking in the DNA was compared with its loss of template activity. Interstrand crosslinks were gradually eliminated from alkylated T, DNA by incubation, under sterile conditions, for variable times at 37 "C; these DNAs were then used as templates for RNA synthesis in vitro; the antibiotic, rifampicin, was included in the synthesis medium to eliminate non-specific chain initiation. T, DNA was alkylated with radioactive nitrogen mustard to calculate the extent of alkylation a t the dose which reduces activity to 3701, of the control.Template activity decreases with increasing doses of nitrogen mustard ; however, the fraction of DNA molecules remaining without interstrand crosslinks decreases much more rapidly. Since only 20°/, of T, DNA is transcribed by E. coli RNA polymerase, we examined the relation between the fraction of molecules bearing one interstrand crosslink in this region and the inhibition of RNA synthesis. The inhibition is significantly greater ; interstrand orosslinks could, a t the most, explain only one third of the activity loss.As interstrand crosslinks disappear from alkylated T, DNA, its template activity, in the presence of rifampicin, decreases. A significant but smaller loss of activity (39°/0 after 72 h of incubation) is observed in the non-alkylated control DNA. When RNA synthesis is carried out in the absence of rifampicin, the activity losses of the control and of the alkylated sample are entirely comparable. Had interstrand crosslinks been responsible for the initial loss of activity after alkylation, their disappearance should have caused a reactivation ; had they been without influence, activity would have been expected to remain constant. We tentatively interpret our result to indicate that other phenomena, i.e. depurination and single strand breakage, which occur during the incubation of alkylated DNA, are responsible for the loss of activity; the possible effect of interstrand crosslinks may thus be masked.At the concentration of nitrogen mustard which reduces DNA template activity to 37O/, of the control, T, DNA is alkylated to the extent of 90 alkyl groups per molecule. This dose is about 9 times less than that necessary with methylation or ethylation of T,...

The Effects of DNA Covalent Adducts on in Vitro Transcription

“…The elongation stops can generally be ascribed to inter- and intrastrand cross-links, although some covalent lesions may be bypassed by the bacterial enzyme . These results support the early hypothesis that nitrogen mustard intrastrand and interstrand cross-links are the major lesions responsible for in vitro transcription inhibition .…”

“…Different covalent adducts inhibit RNA synthesis to a different extent. While covalent binding of 0.2−0.8 drug molecules of nitracrine, furocoumarin, , or other large aromatic compounds , per 10 3 bases are sufficient to reduce RNA synthesis to 37%, a higher degree of template modification by nitrogen mustard (1.1 drug molecules per 10 3 bases) or methylating , and ethylating 94 agents (10−50 modified bases per 10 3 bases) is required in order to achieve the same result. Generally, bulky adducts inhibit RNA synthesis to a greater extent than small alkylating species.…”

“…c Interpolated from two experimental points. d Data from refs and . Other data were calculated for this review.…”

“…The former reacts mainly with N7 of G ( 18 ) and, to a lesser extent, N3 of A, while the latter causes ethylation of the oxygen atoms of G or phosphate residues . However, DNA depurination occurring at the alkylation sites complicates the evaluation of data. , More recent experiments lead to the conclusion that N3 methylation of A affects T7 RNA pol although E. coli RNA pol is less sensitive . Both initiation and total RNA synthesis are reduced to a similar extent, and no appreciable changes in RNA chain length are observed at low enzyme/template ratio.…”

“…160 These results support the early hypothesis that nitrogen mustard intrastrand and interstrand cross-links are the major lesions responsible for in vitro transcription inhibition. 100 When the reaction is continued for up to several hours, the modified bases are hydrolyzed, and this in turn releases the transcription blockages. 144 The stops which are persistent under these conditions appear to correspond to interstand cross-links occurring between purines in the G-N-C/G-N-C or G-N-T/A-N-C sequences 144 (see also note 32).…”