Influence of free ubiquitin concentration and validation results on doa4 Δ background yeast cells at 20 J/m2 UV dose. The figure shows the simulated dynamics of PCNA mono-ubiquitylation (A) and poly-ubiquitylation (B) at a UV dose of 20 J/m2, obtained from a PSA executed on the initial amount of ubiquitin, which is varied in the interval [870, 17396] molecules – mimicking the biological conditions ranging from a 10-fold reduction (corresponding to the severely impaired condition of doa1 δ yeast cells) to a 2-fold overexpression of the total amount of free ubiquitin in WT cells (see the reference value in Table 2). In the plots, the thick lines correspond to the dynamics obtained with the reference value for ubiquitin amount. The simulations show that for ubiquitin amounts lower than the reference value, the amounts of mono- and poly-ubiquitylated PCNA decrease, as also observed experimentally in doa4 δ cells (C, right part). On the other hand, by increasing the ubiquitin amount occurring in the system, the dynamics show an initial peak in the amount of mono- and poly-ubiquitylated PCNA, suggesting that high amounts of ubiquitin might lead the system to a faster bypass of all lesions with respect to the physiological reference value. (C) Western blot showing a comparison between time-course measurements in WT yeast cells (left part) and doa4 δ yeast cells (right part) of the mono-, di- and tri-ubiquitylated PCNA isoforms (top part, denoted by α-Ub) and of non modified PCNA (bottom part, denoted by α-His), sampled at 30 min after UV irradiation. As the aim of these experiments was not to carry out a precise quantification of the PCNA ubiquitylated isoforms, but only to verify the prediction of computational analysis, they were conducted with a single repetition.