Abstract:
Errors in DNA replication and repair can result in mutations, which can lead to cell death, cancer and other genetic diseases. Mutational hotspots include quasipalindromes, which are imperfect inverted repeats that are mutated through a template-switch event, and tandem repeats which are prone to expansion and deletion. Using reporters engineered into the Escherichia coli (E. coli) lacZ gene, we sought to determine which compounds stimulate simple mutations, like base substitutions, and more complex ones, involving template switching and tandem repeats. Our large-scale disk diffusion screen was effective in discovering eleven replication inhibitors that induce template switching, including azidothymidine and ten types of fluoroquinolone antibiotics. We adapted our screen to investigate the mutator effect of four replication inhibitors, 5-azacytidine, formaldehyde, ciprofloxacin, and azidothymidine on simple mutations and mutational hotspots. We identified seven types of mutations, including template switching, tandem repeats, and a base substitution, that were stimulated by one or more of these compounds. Using mutation assays, we quantified the mutational stimulation of formaldehyde and 5-azacytidine on template switching, a tandem repeat, and a base substitution. Since 5-azacytidine inhibits the only cytosine methyltransferase in E. coli, Dcm, we evaluated the Dcm-dependence of these three 5-azacytidine stimulated mutations. After investigating short tandem repeats, we decided to investigate long tandem repeats (LTRs) and successfully engineered LTR deletion reporters into the lacZ gene of the E. coli chromosome. Overall, our methods effectively identified mutagens that stimulate mutations of interest in E. coli, improving our knowledge and expanding our toolset available to explore mutagenesis.
