Ubiquitin-dependent DNA damage bypass is separable from genome replication

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Abstract
The PCNA (proliferating cell nuclear antigen) clamp encircles DNA and thus tethers polymerases to DNA during replication. Ubiquitination of PCNA after DNA damage, which is mediated by proteins of the Rad6 pathway of post-replicative repair (PRR), facilitates DNA damage bypass by dictating which polymerase is recruited to the fork. When this occurs has been a topic of much debate. Daigaku et al. now show that PRR can be postponed until much of the undamaged genome is replicated. The experimental system also allows them to conclude that PRR of DNA lesions occurs mainly by an error-prone process, with error-free bypass playing a minor role. Post-replicative repair (PRR) enables cells to bypass or overcome DNA damage during DNA replication. In eukaryotes, ubiquitylation of the replication clamp PCNA by components of the RAD6 pathway activates damage bypass. When this occurs has been debated. It is now shown that PRR can be postponed until much of the undamaged genome is replicated. Moreover, it seems that PRR occurs mainly by an error-prone process, with error-free bypass playing a minor role. Post-replication 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 polymerases3,4,5, polyubiquitylation is required for an error-free pathway that probably 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 characterized. Here we demonstrate that the RAD6 pathway normally operates during S phase. However, using an inducible system of DNA damage bypass in budding yeast (Saccharomyces cerevisiae), we show that the process is separable in time and space from genome replication, thus allowing direct visualization 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 revealed the distribution of PRR tracts in a synchronized 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.