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Host-cell reactivation of alkylated T7 bacteriophage
Abstract:
Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.
Insights
Alkylated T7 phage survival depends on bacterial DNA repair. Base excision repair, particularly DNA polymerase I activity, is crucial for repairing phage DNA damage caused by methyl methanesulfonate.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Methyl methanesulfonate (MMS) induces DNA alkylation, a form of damage.
- Escherichia coli possesses a base excision repair (BER) pathway to counteract DNA damage.
- T7 phage DNA is susceptible to alkylation and subsequent repair mechanisms.
Purpose of the Study:
- To investigate the role of the base excision repair pathway in repairing alkylated T7 phage DNA.
- To identify specific DNA repair proteins involved in T7 phage survival after MMS treatment.
Main Methods:
- Purified T7 phage was treated with methyl methanesulfonate.
- Phage survival was assayed on various Escherichia coli K-12 host strains with deficiencies in BER pathway components.
- Phage survival was measured immediately after alkylation and after incubation to allow depurination.
Main Results:
- Phage survival was lowest in a mutant lacking DNA polymerase I polymerase activity.
- Strains deficient in apurinic site endonuclease or DNA polymerase I exonuclease activity showed higher phage survival.
- Wild-type strains exhibited the highest survival rates for alkylated T7 phage.
Conclusions:
- Alkylated T7 phage is repaired through the base excision repair pathway in Escherichia coli.
- DNA polymerase I, including its polymerase and exonuclease activities, plays a significant role in this repair process.