Real time dynamics of mutagenesis reveal the chronology of DNA repair and damage tolerance responses in single cells
Significance A central goal in genetics is to understand how mutation rates are regulated by the genome maintenance system in response to DNA damage or drug treatments. This has been challenging because existing mutation assays only show time and population averages of mutation rates and do not resolve the underlying molecular processes. Toward this goal, I utilized a microscopy based method which enables relating the creation of DNA mismatches to single cell gene expression dynamics in real time. I show that DNA alkylation damage causes a distinct pulse of mutagenesis that is shaped by the chronology of constitutive and inducible DNA repair and damage tolerance pathways. Stochastic fluctuations in the expression of these pathways modulated the dynamics of mutagenesis in single Escherichia coli cells. Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy based approach revealed the real time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell to cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage.
Proceedings of the National Academy of Sciences