Visualising the regulation of bacterial DNA repair and mutagenesis using single-molecule and single-cell microscopy

The accurate detection and repair of DNA damage is crucial for genome stability in all organisms. Despite extensive characterization of DNA repair pathways using genetics and biochemical assays, it remains unclear how repair proteins perform their function within the cellular environment and how the different repair pathways are coordinated. I will present our developments of single-molecule imaging and microfluidics techniques to measure DNA repair and mutagenesis in single bacterial cells. By combining super-resolution localisation microscopy and single-molecule tracking, we were able to directly follow the movement of repair enzymes, and obtained new insight into lesion recognition and kinetics of DNA excision repair pathways.

A key advantage of single-cell and single-molecule techniques is their ability to resolve biological heterogeneity and dynamic behaviour without ensemble averaging. Using these approaches, we found that stochasticity, or "noise", can play an important role in the function of the DNA repair system. By imaging the expression of DNA repair proteins in single cells, we discovered that the activation of a transcriptional response to DNA alkylation damage was highly stochastic. The resulting transient lack of repair capacity increased mutation rates in a subpopulation of cells. Random phenotypic variation can therefore lead to heritable genetic changes. Building on these findings, we show how mutation rates are dynamically regulated by the activity of alternative DNA damage and stress responses.