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Mutations induced by methylene blue plus light in single-stranded M13mp2
T J McBride1, J E Schneider, R A Floyd
1Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Abstract:
Reactive oxygen species are generated by a variety of cellular processes. These endogenously generated, reactive intermediates produce a multiplicity of DNA alterations and mutations and have been implicated in the pathogenesis of several human diseases. We report here that treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light generates increased levels of 8-hydroxydeoxyguanosine and that mutagenesis is both highly specific and dependent on the SOS response. Lesions produced block the progression of DNA synthesis one base preceding template guanines. In SOS-induced Escherichia coli, 97% of all methylene blue-induced mutations in the lacZ alpha gene of M13mp2 DNA are single-base substitutions opposite template guanines. The most frequent mutations are G----C transversions. The G----T transversions expected from the presence of 8-hydroxydeoxyguanosine in the template strand occur, but at a lower frequency. Sequence data together with SOS dependency and the presence of replication blockage demonstrate that while 8-hydroxydeoxyguanosine may serve as an important marker to monitor oxygen-induced DNA damage in humans, it does not account for either the observed blockage to replication or the mutagenesis by methylene blue plus light in SOS-induced E. coli. Instead, an as yet unidentified lesion generated by active oxygen species is a more potent mutagenic event.
Insights
Methylene blue and light generate DNA damage, causing mutations in E. coli. While 8-hydroxydeoxyguanosine is a marker, an unknown lesion is the primary mutagenic cause.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Reactive oxygen species (ROS) are byproducts of cellular metabolism.
- ROS can cause DNA damage, leading to mutations and diseases.
- Methylene blue (MB) plus light is a known photosensitizer that generates ROS.
Purpose of the Study:
- To investigate the DNA damage and mutagenesis induced by methylene blue and white light.
- To determine the role of 8-hydroxydeoxyguanosine (8-OHdG) in MB-induced mutagenesis.
- To identify the specific DNA lesions responsible for replication blockage and mutations.
Main Methods:
- Treatment of single-stranded M13mp2 bacteriophage DNA with methylene blue and white light.
- Quantification of 8-hydroxydeoxyguanosine (8-OHdG) levels.
- Analysis of mutations in the lacZ alpha gene of M13mp2 DNA in SOS-induced Escherichia coli.
- DNA sequencing to identify mutation types and locations.
Main Results:
- Methylene blue and white light treatment increased 8-OHdG levels in DNA.
- Mutagenesis was highly specific, SOS-dependent, and primarily caused G----C transversions.
- Replication blockage occurred one base before template guanines, and 8-OHdG did not fully explain the observed mutations or blockage.
- An unidentified lesion, not 8-OHdG, was identified as the primary mutagenic event.
Conclusions:
- 8-hydroxydeoxyguanosine is a marker for oxygen-induced DNA damage but not the sole cause of MB-induced mutagenesis or replication blockage.
- An unknown DNA lesion generated by ROS is a more potent mutagenic event than 8-OHdG.
- Further research is needed to identify the specific lesion responsible for MB-induced mutagenesis.