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SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light
1LA147 CNRS, U140 INSERM, Groupe Réparation des lésions radio et chimioinduites, Institut Gustave Roussy, Villejuif, France.
Abstract:
In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mp18 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvr A fpg-1 double mutant than that seen in uvr A, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GC-->TA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly G-->T transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.
Insights
Methylene blue and light (MB-light) cause DNA damage, leading to mutations in bacterial cells. This DNA mutagenesis is partly independent of the SOS response and may involve 8-oxo-7,8-dihydroguanine lesions.
Area of Science:
- Molecular Biology
- Genetics
- Photochemistry
Background:
- Visible light in the presence of methylene blue (MB-light) induces DNA lesions.
- Understanding the mutagenic potential of these lesions is crucial for assessing DNA damage and repair mechanisms.
Purpose of the Study:
- To analyze the lethal and mutagenic potential of MB-light-induced DNA lesions.
- To investigate the role of bacterial DNA repair pathways, including SOS functions, in MB-light mutagenesis.
- To determine the types of mutations induced by MB-light treatment in both double-stranded (ds) and single-stranded (ss) M13mp18 DNA.
Main Methods:
- In vitro photosensitization of M13mp18 DNA using methylene blue and visible light.
- Transfection of treated DNA into various bacterial hosts (wild-type, uvrA, fpg-1, umuC mutants).
- Forward mutation assay targeting the lacZ gene for mutagenesis determination.
- DNA sequencing to analyze mutation spectra.
Main Results:
- MB-light treatment of ds M13mp18 DNA increased mutation frequency fivefold in wild-type cells at 10% survival.
- Mutagenesis was higher in the uvrA fpg-1 double mutant compared to single mutants or wild-type.
- Sequence analysis revealed predominantly single base substitutions (GC-->TA) in wild-type for ds DNA and G-->T transversions for ss DNA.
- MB-light-induced mutagenesis is partially independent of SOS functions in Escherichia coli.
Conclusions:
- MB-light induces significant mutagenesis in bacterial DNA, with distinct mutation patterns for ds and ss DNA.
- Bacterial repair systems, particularly those involving uvrA and fpg-1, play a role in processing MB-light-induced DNA lesions.
- The results suggest 8-oxo-7,8-dihydroguanine as a major promutagenic lesion, highlighting its significance in MB-light-induced DNA damage.