Timing and spacing of ubiquitin‐dependent DNA damage bypass

Abstract

DNA damage tolerance, also called postreplication repair (PRR), is a pathway that enables cells to overcome replication problems and complete the duplication of their genomes in the presence of DNA lesions. In eukaryotes, the pathway is controlled by the ubiquitylation of the replication clamp protein PCNA. Monoubiquitylation of PCNA activates translesion synthesis by specialized damage‐tolerant DNA polymerases, whereas polyubiquitylation is required for an alternative, error‐free pathway. Ubiquitin‐dependent PRR substantially influences the accuracy of replication and the resistance towards genotoxic agents; however, the details of when during the cell cycle the pathway is most relevant remain a matter of debate. We have now designed an inducible system of damage bypass in budding yeast that has allowed us to analyze the effects of PRR in the course of the cell cycle and to separate its activity from bulk replicative DNA synthesis. We show that PRR can act not only during S phase, but similarly contributes to survival when activated after replication of the genome is largely complete. We find that PRR both during and after S phase is largely mediated by translesion synthesis, with error‐free damage avoidance acting as a back‐up pathway. Finally, we have directly visualized PRR tracts by fiber spreading.