Asymmetric down-regulation of tolerance to DNA damage at replication forks

The tolerance pathway allows bypassing deleterious lesions that block replicative DNA polymerases. In eukaryotes tolerance is controlled by ubiquitylation of the DNA replication factor PCNA4. Here, we show that PCNAK164-ubiquitin proteases (PCNA-DUBs) Ubp10 and Ubp12 localise to replication forks. In particular, the main PCNA-DUB, Ubp10, associates primarily to nascent lagging strands. We also found that cells deficient for PCNA deubiquitylation show both increased interaction of TLS polymerase ζ-associated Rev1 with lagging strands and Rad52-dependent template switching events. This evidence reveals a fork-coupled layer of DDT regulation allowing strand-specific pathway choices. Chip on chip analysis was carried out as described (Bermejo et al., 2011), employing anti-myc monoclonal antibody clone 4A6 (Millipore) Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.