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Effect of abasic sites on bacteriophage T7 protein synthesis
G Sanchez1, J F Racine, M D Mamet-Bratley
1Département de biochimie, Université de Montréal, Quebec, Canada.
Abstract:
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.
Insights
Methyl methanesulfonate alkylation of bacteriophage T7 DNA creates apurinic sites. These sites inhibit protein synthesis and transcription in Escherichia coli, with weaker effects on transcription than replication.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA damage, specifically apurinic sites, can disrupt essential cellular processes.
- Bacteriophage T7 serves as a model system for studying DNA replication and transcription in vivo.
- Escherichia coli possesses DNA repair mechanisms, including the xth nfo gene products, to counteract DNA damage.
Purpose of the Study:
- To investigate the impact of in vivo induced apurinic sites in bacteriophage T7 DNA on protein synthesis.
- To compare the effects of these apurinic sites on transcription and replication.
- To elucidate the role of DNA repair proficiency in mitigating the consequences of apurinic sites.
Main Methods:
- Alkylation of bacteriophage T7 DNA with methyl methanesulfonate to generate apurinic sites.
- Infection of both repair-proficient and repair-deficient (xth nfo mutant) Escherichia coli strains with modified phage.
- Analysis of bacteriophage protein synthesis classes (I, II, and III) via in vivo assays.
Main Results:
- In repair-proficient E. coli, all three classes of T7 proteins were synthesized, but with significant delays.
- In repair-deficient E. coli, only class I proteins were produced, with delayed and reduced synthesis.
- These findings indicate that apurinic sites inhibit T7 protein synthesis and transcription in vivo.
Conclusions:
- Apurinic sites in bacteriophage T7 DNA impede transcription and protein synthesis in Escherichia coli.
- The observed effects on transcription are less pronounced than those on DNA replication.
- DNA repair mechanisms influence the cellular response to apurinic site-induced damage.