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Mechanism of toxicity of 3-methyladenine for bacteriophage T7
J F Racine1, Y Zhu, M D Mamet-Bratley
1Département de Biochimie, Université de Montréal, Qué., Canada.
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.
Insights
Methyl methanesulfonate damages bacteriophage T7 DNA, inhibiting T7 RNA polymerase transcription in repair-deficient Escherichia coli. This DNA damage specifically hinders the elongation step of transcription, impacting phage gene expression.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Methyl methanesulfonate (MMS) is an alkylating agent that causes DNA damage.
- Bacteriophage T7 gene expression is regulated by its own RNA polymerase.
- DNA repair mechanisms in Escherichia coli are crucial for maintaining genome integrity.
Purpose of the Study:
- To investigate the effect of methyl methanesulfonate-induced DNA damage on bacteriophage T7 gene expression.
- To determine the impact of unrepaired DNA lesions on T7 RNA polymerase activity.
- To elucidate the mechanism by which DNA alkylation affects T7 transcription.
Main Methods:
- Treatment of bacteriophage T7 with methyl methanesulfonate.
- Infection of repair-proficient and repair-deficient Escherichia coli strains.
- Analysis of phage protein synthesis.
- In vitro transcription assays using alkylated T7 DNA and purified T7 RNA polymerase.
- Characterization of transcription initiation and elongation steps.
Main Results:
- In repair-deficient cells, MMS-treated phage T7 synthesized only early proteins, indicating inhibition of late transcription.
- T7 RNA polymerase activity was significantly reduced when transcribing from alkylated DNA templates.
- In vitro assays showed that alkylation hindered both chain initiation and elongation, with a more pronounced effect on elongation (translocation).
Conclusions:
- 3-methyladenine lesions, a product of MMS, exert toxicity at the transcriptional level by inhibiting T7 RNA polymerase.
- T7 RNA polymerase is highly sensitive to DNA alkylation, more so than E. coli RNA polymerase.
- The primary mechanism of inhibition involves hindering the translocation of T7 RNA polymerase along the damaged DNA template during elongation.