Ubiquitin-dependent DNA damage bypass is separable from genome replication

Postreplication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication1. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway2. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases3-5, polyubiquitylation is required for an error-free pathway that likely involves a template switch to the undamaged sister chromatid6. Both the timing of PRR events during the cell cycle and their location relative to replication forks, as well as the factors required downstream of PCNA ubiquitylation, have remained poorly characterised. Here we demonstrate that the RAD6 pathway normally operates during S phase. However, using an inducible system of DNA damage bypass in budding yeast, we show that the process is separable in time and space from genome replication, thus allowing direct visualisation and quantification of productive PRR tracts. We found that both during and after S phase ultraviolet radiation-induced lesions are bypassed predominantly via translesion synthesis, whereas the error-free pathway functions as a backup system. Our approach has for the first time revealed the distribution of PRR tracts in a synchronised cell population. It will allow an in-depth mechanistic analysis of how cells manage the processing of lesions to their genomes during and after replication.