Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28. Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo. We also demonstrated that Eco1 acetylated polymerase η in vitro. Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.

The SMC complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in S. pombe, we hypothesized that active transcription facilitates damage-induced cohesion in S. cerevisiae. Here, we found that expression of genes involved in chromatin assembly and positive transcription regulation were relatively enriched in WT compared to Polη-deficient cells (rad30Δ). The rad30Δ mutant showed a dysregulated transcriptional response and increased cohesin binding around transcription start sites. Perturbing histone exchange at promoters adversely affected damage-induced cohesion, similarly to deletion of RAD30. Conversely, altering chromatin accessibility or regulation of transcription elongation, suppressed the lack of damage-induced cohesion in rad30Δ cells. These results indicate that Polη promotes damage-induced cohesion through its role in transcription, and support the model that regulated transcription facilitates formation of damage-induced cohesion.

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in the Polη null mutant (rad30Δ), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30Δ compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30Δ cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30Δ cells. Furthermore, transcription inhibition negatively affected formation of damage-induced cohesion. These results indicate that the transcriptional deregulation of the Polη null mutant is connected with its reduced capacity to establish damage-induced cohesion. This also suggests a linkage between regulation of transcription and formation of damage-induced cohesion after replication.

It has been found that calorie restriction (CR) can extend the average and maximum life span from yeast to primates and delay the onset of age‐associated pathologies. Mismatch repair and double strand break repair systems are major DNA repair pathways whose function are critical for maintaining genome stability. The inactivation of these systems in human pathway has been reported to cause cancer predisposition such as Hereditary Nonpolyposis Colorectal Cancer, Werner and Bloom syndromes. By using Saccharomyces cerevisiae model system for CR and aging studies, DNA repair defective cells (msh2Δ, msh3Δ, msh6Δ and sgs1Δ) were cultured with normal (2% glucose) and CR (0.5% glucose) medium. Genome stability studies were analyzed by CANr, Homr and Lysr mutation assay. Results show that CR can significantly extend the lifespan of all wild‐type and mutants. Besides, CR shows significant decrease in HOMr mutation frequency by 2.7 × 104‐fold in msh2Δ mutant, whereas by 2.4 × 106‐fold and 1.4 × 106‐fold reduction in msh3Δ mutant for HOMr and LYSr assay respectively. In addition, for sgs1Δ mutant, there are significant decreases in HOMr and LYSr assay by two‐fold. Under normal condition, msh2Δ, msh3Δ and sgs1Δ cells found to have regrow phenotype upon aging. However, this phenotype was diminished under CR condition. Thus, CR promotes genome stability in DNA repair defective cells especially in msh2Δ, msh3Δ and sgs1Δ mutants.