Understanding the role and regulation of Polη for damage-induced cohesion

Abstract: In response to DSBs, de novo sister chromatid cohesion can be established after DNA replication in Saccharomyces cerevisiae. The damage-induced cohesion is formed close to the break and genome-wide on undamaged chromosomes. Polymerase eta (Polη), which bypasses ultraviolet-induced DNA lesions, is specifically required for genome-wide damage-induced cohesion. However, its role in this process is unclear. The works in this thesis aimed to understand the mechanistic role of Polη for damage-induced cohesion, and how damage-induced cohesion is generated genome-wide. In the first study, the possibility that Cdc28 and Eco1 modulate activity of Polη was being investigated. Based on in vitro kinase assay and structure modeling, Polη appeared to be an attractive substrate for the cyclin-dependent kinase, Cdc28. Abolishing the potential Polη-S14-phosphorylation by a serine to alanine mutation resulted in reduced protein level of Polη and impaired damage-induced cohesion in vivo. Although Polη was acetylated by the acetyltransferase Eco1 in vitro, absence of Eco1 did not affect protein level or nuclear accumulation of Polη in vivo. This contrasted with certain non-acetylatable Polη-KR mutants, implicating that other factor could regulate Polη through modifying these lysine residues. In the second study, it was hypothesized that transcriptional activation/regulation facilitates generation of damage-induced cohesion, based on related findings in budding yeast and fission yeast. Absence of Polη or prevention of Polη-S14-phosphorylation perturbed transcription elongation because the mutants were sensitive to transcription elongation inhibitors and showed reduction of RNA polymerase II-binding on chromatin. Therefore, mutants defective in histone exchange or regulation of transcription elongation were created to mimic or suppress the transcriptional deficiency of the Polη null mutant (rad30Δ). Interestingly, these mutants mimicked or suppressed the lack of damage-induced cohesion in rad30Δ cells. Furthermore, establishment of damage-induced cohesion was compromised by transcription inhibition. Taken together, these studies showed that Polη is potentially phosphorylated by Cdc28 at the S14 residue, which appeared to be functionally related with transcription and formation of damage-induced cohesion. In addition, persistent absence of Polη affects transcriptional regulation. This consequently impaired formation of damage-induced cohesion, implicating a linkage between transcriptional regulation and establishment of genome-wide damage-induced cohesion after DNA replication.