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Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome
1A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Abstract:
The spectrum of mutations induced upon in vivo replication of an M13 genome containing a site-specifically located propanodeoxyguanosine (PdG) adduct was determined. PdG was used as a model for the major deoxyguanosine adduct produced on reaction of DNA with the endogenous genotoxin malondialdehyde. PdG was introduced at position 6256 of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(PdG)TCCG-3' into an 8-base gap in the (-)-strand of duplex M13MB102. Replication of the adducted strand was maximized by incorporation of uracil into the unadducted (+)-strand. Following replication of dG-containing and PdG-containing M13MB102 genomes in Escherichia coli JM105, frameshift mutations were detected as phenotypic changes in the lacZ alpha marker gene. Base pair substitutions were detected by differential hybridization using 32P-labeled 13-mers bearing different bases opposite position 6256. Neither frameshift nor base pair substitution mutations were detected following replication of PdG-adducted genomes in non-SOS-induced JM105. However, PdG-->T transversions and PdG-->A transitions were detected following transformation of PdG-adducted M13MB102 into SOS-induced JM105. Both types of mutations were detected at comparable frequencies, and the total mutation frequency was approximately 2%. The results indicate that PdG is an efficient premutagenic lesion in E. coli strains in which the SOS response is induced.
Insights
Propanodeoxyguanosine (PdG) adducts in M13 DNA cause mutations in SOS-induced Escherichia coli. Specifically, PdG leads to transversions and transitions, acting as a premutagenic lesion.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Malondialdehyde is an endogenous genotoxin that forms deoxyguanosine adducts in DNA.
- Propanodeoxyguanosine (PdG) is a major adduct formed by malondialdehyde.
- Understanding the mutagenicity of such adducts is crucial for assessing DNA damage and repair mechanisms.
Purpose of the Study:
- To determine the spectrum of mutations induced by a site-specific propanodeoxyguanosine (PdG) adduct during in vivo DNA replication.
- To investigate the role of the SOS response in the mutagenic potential of PdG adducts.
- To model the genotoxic effects of malondialdehyde-induced DNA damage.
Main Methods:
- Site-specific incorporation of a PdG adduct into the M13 genome.
- Replication of the adducted M13 genome in Escherichia coli strains, both with and without SOS induction.
- Detection of frameshift mutations via phenotypic changes in the lacZ alpha marker gene.
- Detection of base pair substitutions using differential hybridization.
Main Results:
- No mutations were detected in non-SOS-induced E. coli.
- In SOS-induced E. coli, PdG adducts resulted in PdG to thymine transversions and PdG to adenine transitions.
- Both mutation types occurred at comparable frequencies, with a total mutation frequency of approximately 2%.
Conclusions:
- Propanodeoxyguanosine (PdG) is an efficient premutagenic lesion in E. coli.
- The SOS response is essential for the mutagenicity of PdG adducts.
- These findings highlight the genotoxic potential of endogenous aldehydes and their DNA adducts.