Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Mitogen-activated protein kinase (MAPK) cascades are conserved signalling modules that control many cellular processes by integrating intra-and extracellular cues. The p38/Hog1 MAPK is transiently activated in response to osmotic stress, leading to rapid translocation into the nucleus and induction of a specific transcriptional program. When investigating the dynamic interplay between Hog1 activation and Hog1-driven gene expression, we found that Hog1 activation increases linearly with stimulus, whereas the transcriptional output is bimodal. Modelling predictions, corroborated by single cell experiments, established that a slow stochastic transition from a repressed to an activated transcriptional state in conjunction with transient Hog1 activation generates this behaviour. Together, these findings provide a molecular mechanism by which a cell can impose a transcriptional threshold in response to a linear signalling behaviour. The authors declare that they have no competing financial interests. Transcriptional activation of mating genes occurs with linear kinetics and high fidelity (5,6), and the observed cell-to-cell variation in protein expression is governed by the ability of cells to express proteins (expression capacity) (5). While the mating pathway can be compared to a cell-fate decision system with sustained MAPK activity, the HOG pathway is an adaptation response, which is only transiently induced like other stress-activated pathways (7). We therefore investigated whether this transient response would trigger different expression behaviour. Mitogen-activated protein kinase (MAPKTo quantitatively measure the transcriptional output induced by osmotic stress, we engineered a reporter system based on a quadruple Venus (qV) fluorescent protein expressed under the control of specific osmo-stress-inducible promoters dependant on the three main transcription factors orchestrating the transcriptional response to osmotic 3 stress (Hot1 and Sko1: pSTL1, Msn2,4: pALD3 or Msn2,4 and Hot1: pHSP12) (8). Flow cytometry revealed a Pbs2-dependent 20-fold increase in pSTL1-qV reporter expression when 0.4M NaCl was added to the growth medium ( Fig. 1 A and B). also generated a bimodal expression output of the Ste12-specific reporter pFIG1-qV.However, signalling in the mating pathway is prevented from "Start" through S phase (9), and expression output became unimodal after relieving this cell-cycle dependent restriction ( Fig. 1B and Fig. S1B).To investigate the source of the HOG pathway bimodal expression behaviour, we integrated two reporters driving the expression of a quadruple cyan fluorescent protein (qCFP) and a qV construct in the same cell. Correlation of the cyan and yellow intensities measures the contribution of cell-to-cell (extrinsic) and intra-cellular (intrinsic) variability to the overall expression noise (5, 10). The two pFIG1-reporters induced by !-factor demonstrated that the mating pathway is governed by extrinsic noise. In contrast,we observed a lack of correlation between the two pSTL1-repor...