Margaret D. Mamet-Bratley

Alkylated DNA as template in the synthesis of RNA in vitro

Abstract T7 DNA, alkylated by methyl or ethyl methane sulfonate under conditions where depurination and single-strand breakage are minimized, shows reduced template activity for RNA synthesis in vitro. The inactivation produced by alkylation appears to be a single-hit process. At the 37 % activity level, there are about 800 alkyl groups per DNA; the sensitive region of the DNA thus contains about 50 nucleotide pairs and could be the region containing the initiation site for mRNA corresponding to the early phage proteins. Alkylation per se of DNA and the subsequent inactivation of its template activity may explain a small part of the immediate inactivation of bacteriophages caused by alkylating agents.

A new electron microscopic method for studying protein-nucleic acid interactions.

Abstract A new method for preparation of nucleic acid specimens for electron microscopy has been adapted to study the interaction of proteins with DNA. Both a detergent and a basic protein are added to the DNA-protein solution before spreading on a hypophase containing 0.2 m ammonium acetate. This method has been tested using T7 DNA and Escherichia coli RNA polymerase. Specifically bound enzyme molecules were clearly visible on the well extended DNA molecules; the binding sites were located at 0.59, 1.24, 1.57, and 1.86% of the total length of T7 DNA. Under carefully controlled conditions, 40–85% of the DNA molecules specifically bound at least one enzyme molecule.

Initiation by RNA polymerase on alkylated T7 DNA.

Abstract Alkylation of T7 DNA does not create new initiation sites for Escherichia coli RNA polymerase.

Mechanism of toxicity of 3-methyladenine for bacteriophage T7.

Treatment of bacteriophage T7 with methyl methanesulfonate perturbed phage-specific genetic expression in both repair-proficient and repair-deficient Escherichia coli cells. In wild-type cells (AB1157), the time course of protein synthesis was slowed down but an entire complement of phage proteins was synthesized. In cells (BK2114, tag-) unable to repair 3-methyladenine, the toxic lesion produced by methyl methanesulfonate, alkylated phage produced only early (class I) proteins. These results suggested that late transcription was inhibited in infected tag- cells. These cells were shown to contain a significant amount of active T7 RNA polymerase, a class I protein. Thus, the cause of inhibition appeared to be the inability of T7 RNA polymerase to use unrepaired DNA as template. In vitro transcription assays with alkylated T7 DNA as template supported this proposal. T7 RNA polymerase proved to be very sensitive to the presence of alkylation lesions. In addition, the phage enzyme was much more sensitive to these lesions than was its bacterial counterpart, E. coli RNA polymerase. These results suggest that 3-methyladenine exerts its toxic action, in the T7 system, at the level of transcription by T7 RNA polymerase. To further characterize the reduced activity of the T7 enzyme, an in vitro transcription assay using linearized plasmid DNA with one T7 promoter was devised. Gel electrophoresis revealed that only one transcript of well-defined length was synthesized by T7 RNA polymerase on this template. Alkylation of the template did not alter the size of the transcript produced. Simultaneous measurement of chain initiation and chain elongation confirmed this result by showing that both steps were reduced to the same extent by alkylation of template DNA. Thus T7 RNA polymerase does not appear to be blocked by 3-methyladenine. Rather the lesion must hinder translocation of T7 RNA polymerase along the DNA template during chain elongation.

Transcription in vitro from a DNA template containing apurinic sites

Abstract T7 DNA containing a limited number of apurinic sites shows reduced template activity for RNA synthesis in vitro. Initiation specificity remains unchanged by the presence of apurinic sites. Quantitative analysis shows that depurination is much more detrimental than alkylation to the template activity of DNA. Such a result is in accord with phage-lethality studies which show, for monofunctional alkylating agents, that depurination is much more toxic than alkylation.

The molecular weight of a rabbit antibody

Abstract Rabbit antibody, specific for the 2,4-dinitrophenyl group attached to lysine, exhibits a weak tendency to associate at pH 7.4, 20°; its monomer molecular weight appears to be 135 000 ± 5000. These conclusions are supported by detailed physicochemical studies of the antibody. At protein concentration below 2 mg/ml, the sedimentation coefficient, s 20, w , increased with protein concentration; at higher concentrations, the s 20, w values showed the more usual decrease with protein concentration but the dependence was abnormally small. Data from sedimentation equilibrium experiments, using Rayleigh optics with 2–3-mm liquid columns of solution, consistently yielded curvature in plots of ln c , protein concentration, versus x 2 , where x is the distance from the center of rotation; heterogeneity was thus indicated. Analysis of these results in terms of the dependance of weight average molecular weight, M w , on concentration showed that M w increased as the protein concentration ( c ) increased. The molecular weight data were adequately described by a theoretical curve of M w versus c , constructed for a dimerizing system with monomer molecular weight 130 000 and association constant 0.04 l/g. The wide scatter of previously reported molecular weight values, 137 000–188 000, can be explained by neglect of the proteins tendency to associate.

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.

Cross-linked DNA as template in the synthesis of RNA in vitro

Abstract T7 DNA cross-linked by limited treatment with HNO2 or nitrogen mustard can serve as template for RNA synthesis in vitro. At low concentrations, HNO2 does not significantly alter the template activity of the DNA. Nitrogen mustard causes a reduction in template activity although DNA molecules with several cross-links are still active for RNA synthesis. Nitrogen mustard, through monofunctional alkylations, can cause single-strand breaks in DNA; such modifications, more frequent than cross-links, could cause the observed activity loss. T7 DNA, highly cross-linked by HNO2, loses much of its activity for RNA synthesis. This observation is in accord with a mechanism for RNA synthesis which involves at least local strand separation in the DNA template.

Difference in the action of ethyl and methyl methane sulfonates on DNA template activity for RNA synthesis in vitro

RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis. Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length. In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.

Effect of abasic sites on bacteriophage T7 protein synthesis

We have examined protein synthesis directed by bacteriophage T7 which had been alkylated with methyl methanesulfonate so as to produce apurinic sites in its DNA in vivo. Both repair-proficient and repair-deficient (xth nfo mutant) strains of Escherichia coli served as host cells. In repair-proficient cells, all three classes of phage proteins were synthesized, although with significant delays. In mutant cells, only class I proteins were produced and their synthesis was delayed and reduced, demonstrating a perturbation of protein synthesis and providing the first in vivo indication that transcription is inhibited by abasic sites. However, the proposed effects of abasic sites on transcription appear to be weaker than those on replication.