Figure 3.

PCNA ubiquitylation is prolonged in pif1Δ and exo1Δ mutants. Ubiquitylation of His₆-tagged PCNA was analysed by Ni-NTA pull-down followed by western blotting in exo1Δ (A), pif1Δ (B) and exo1Δ pif1Δ (C). G1-synchronized cells were treated with 0.04% MMS for 30 min prior to release into S phase without MMS. At the indicated times, samples were collected for isolation of His₆-PCNA under completely denaturing conditions as described in the ‘Materials and Methods’ section. Cell-cycle profiles are shown below the blots. Quantification of polyubiquitylated PCNA, relative to G1 (0 min) and normalized to unmodified PCNA, is plotted on the right. The fold increase in the mutants is indicated above the bars.