Single-mitosis dissection of acute and chronic DNA mutagenesis and repair

The mammalian genome is constantly exposed to genotoxins that can lead to mutations and ultimately tumorigenesis. Despite cancer being recognized as a genetic disease, much is still unknown regarding tumor induction and evolution of the cancer genome. To study gradual (ROS) and acute (UV) mutagenic processes both in vitro and then in vivo, we first use microfluidic separation of cells after a single mitosis following pulse mutagenesis. While UV mutations tend to be fixed after one cell cycle and show mirror image mutation patterns between mitotic sisters, ROS mutations do not. We next show that while both mutation rates are reduced as a function of transcription, only UV has a strand-specific repair phenotype. Lastly, we resolve pulse induced lesion segregation to single haplotypes by analysing liver tumour genomes from F1 mice treated with N-nitroso-diethylamine (DEN). In summary, we have established highly controlled systems to study 3 distinct forms of mutagenesis at their inception in mammalian cells, providing insights into how these mutations are fixed at the genome scale.