Post-replication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required for an error-free pathway that probably involves a template switch to the undamaged sister chromatid. 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.
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