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Repair of methyl methane sulfonate-damaged phage by Haemophilus influenzae
Abstract:
Seven mutants of Haemophilus influenzae strain Rd (mmsA-) have been isolated that are more sensitive to methyl methane sulfonate (mms) than recombination-deficient (recA-) mutants. The mutations cotransformed about 25% with the strA locus while the five studied clustered tightly; they are all probably allelic. The mutants are not sensitive to ultraviolet radiation, X-rays, or nitrous acid. Mms-damaged phage HP1 plated very inefficiently on these mutants, indicating that they lack the first step in the excision repair of the lesion N3-methyladenine (m3A). Incubation of damaged phage at 30 degrees C in the absence of mms resulted in a steady decline of viability when the phage were plated on the wild mmsA+ host but an initial steep rise was seen when it was plated on an mmsA- mutant. The rise is explained by the assumption that m3A lesions hydrolyzed off the DNA giving rise to repairable apurinic sites by both the mmsA+ and mmsA- hosts. No decline in viability was observed when hydroxylamine was present in the medium. This compound is known to prevent or slow down beta-elimination. The delayed decline in viability is therefore explained by assuming that apurinic sites give rise to beta-elimination-induced single strand breaks in the phage DNA that cannot be repaired by either host. Marker rescue experiments indicated that these breaks did not interrupt injection of phage DNA.
Insights
New Haemophilus influenzae mutants (mmsA-) show increased sensitivity to methyl methane sulfonate (mms). These mutants are deficient in repairing N3-methyladenine DNA lesions, impacting phage viability assays.
Area of Science:
- Microbiology
- Molecular Biology
- DNA Repair Mechanisms
Background:
- Methyl methane sulfonate (mms) is a mutagenic agent that causes DNA damage.
- Haemophilus influenzae possesses DNA repair pathways to counteract mutagenic effects.
- Recombination-deficient (recA-) mutants are known to have impaired DNA repair capabilities.
Purpose of the Study:
- To isolate and characterize new mutants of Haemophilus influenzae with enhanced sensitivity to methyl methane sulfonate.
- To investigate the specific DNA repair defect in these mmsA- mutants.
- To elucidate the role of the mmsA gene in the repair of N3-methyladenine lesions.
Main Methods:
- Isolation and genetic characterization of mmsA- mutants in Haemophilus influenzae.
- Complementation analysis and cotransformation experiments to assess allelism.
- Assessing mutant sensitivity to various DNA damaging agents (mms, UV, X-rays, nitrous acid).
- Phage survival assays using mms-damaged phage HP1 plated on wild-type and mutant hosts.
Main Results:
- Seven mmsA- mutants were isolated, exhibiting higher mms sensitivity than recA- mutants.
- Mutations were largely allelic and mapped to a specific locus.
- Mutants showed no increased sensitivity to UV, X-rays, or nitrous acid.
- Mms-damaged phage plated inefficiently on mmsA- mutants, indicating a defect in N3-methyladenine excision repair.
- Phage viability assays revealed a unique response to m3A lesion hydrolysis, suggesting a role in apurinic site formation and subsequent strand breaks.
Conclusions:
- The mmsA gene is crucial for the initial step in the excision repair of N3-methyladenine DNA lesions.
- mmsA- mutants are defective in repairing mms-induced DNA damage, specifically m3A lesions.
- The study provides insights into DNA repair pathways and the consequences of unrepaired DNA damage in bacteria and phage.