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Mechanism of toxicity of 3-methyladenine for bacteriophage T7.
Mutat Res. 1993 Oct; 294(3):285-98.MR

Abstract

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.

Authors+Show Affiliations

Département de Biochimie, Université de Montréal, Qué., Canada.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

7692268

Citation

Racine, J F., et al. "Mechanism of Toxicity of 3-methyladenine for Bacteriophage T7." Mutation Research, vol. 294, no. 3, 1993, pp. 285-98.
Racine JF, Zhu Y, Mamet-Bratley MD. Mechanism of toxicity of 3-methyladenine for bacteriophage T7. Mutat Res. 1993;294(3):285-98.
Racine, J. F., Zhu, Y., & Mamet-Bratley, M. D. (1993). Mechanism of toxicity of 3-methyladenine for bacteriophage T7. Mutation Research, 294(3), 285-98.
Racine JF, Zhu Y, Mamet-Bratley MD. Mechanism of Toxicity of 3-methyladenine for Bacteriophage T7. Mutat Res. 1993;294(3):285-98. PubMed PMID: 7692268.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Mechanism of toxicity of 3-methyladenine for bacteriophage T7. AU - Racine,J F, AU - Zhu,Y, AU - Mamet-Bratley,M D, PY - 1993/10/1/pubmed PY - 1993/10/1/medline PY - 1993/10/1/entrez SP - 285 EP - 98 JF - Mutation research JO - Mutat. Res. VL - 294 IS - 3 N2 - 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. SN - 0027-5107 UR - https://www.unboundmedicine.com/medline/citation/7692268/Mechanism_of_toxicity_of_3_methyladenine_for_bacteriophage_T7_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0921-8777(93)90011-5 DB - PRIME DP - Unbound Medicine ER -