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Mutations in a shuttle vector exposed to activated mitomycin C
N S Srikanth1, A Mudipalli, A E Maccubbin
1Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Abstract:
The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross-links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base- and sequence-specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence. The MMC-induced mutations were predominantly base substitutions. Eighty-four percent of the base substitutions were transversions, with G:C-->T:A the major transversion. Single base deletions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its Rf value) and clustering of MMC-induced mutations at three GC-rich areas (nt 100-123, 152-163, and 168-176) suggested that the mutational spectrum we found was due to binding of MMC to guanine on either strand of the plasmid DNA.
Insights
Mitomycin C (MMC) causes mutations by binding to guanine in DNA. This study reveals that MMC primarily induces G:C to T:A transversions and G:C deletions at specific DNA hotspots.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mitomycin C (MMC) is a potent antibiotic and antitumor agent.
- MMC's cytotoxicity stems from irreversible DNA binding after bioreductive activation.
- MMC alkylating species bind guanine residues, forming DNA monoadducts and cross-links.
Purpose of the Study:
- To investigate the base- and sequence-specific mutations induced by mitomycin C.
- To understand how MMC-induced DNA lesions lead to specific mutational outcomes.
Main Methods:
- Chemical reduction of mitomycin C using sodium borohydride.
- Treatment of shuttle vector pSP189 with reduced MMC.
- Replication of treated DNA in human Ad293 cells.
- Rescue of mutated plasmid DNA in bacteria for sequence analysis.
Main Results:
- MMC induced predominantly base substitutions, with 84% being G:C-->T:A transversions.
- Single base deletions, primarily at G:C sites (77%), were another major mutational event.
- Specific DNA positions (115, 123, 163) were identified as mutational hotspots.
- Mutations clustered in GC-rich regions, suggesting MMC binds to guanine on either DNA strand.
Conclusions:
- The mutational spectrum observed is consistent with mitomycin C binding to guanine residues.
- MMC induces specific types of mutations (G:C-->T:A transversions, G:C deletions) at preferred DNA sequences.