The regulation of dynamic interactions between PARG and PCNA

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Given the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, it has remained unclear how the strength and sequence of their binding to PCNA is regulated. Post-translational protein modifications regulate the dynamics of protein-protein interactions. Post- translational modifications of PCNA - ubiquitination and acetylation - provide a good example of this paradigm: PCNA monoubiquitination elicits polymerase switching, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins by post- translational modifications remains underexplored. Recently we identified a new PCNA-interacting protein motif (PIP-box) within poly(ADP-ribose) glycohydrolase (PARG). PARG is an essential enzyme in eukaryotes that removes poly(ADP-ribose) and is required for replication and recovery from replication stress. We have shown that the PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Furthermore, it appears that PARG itself is also subject to regulation by the same modifications that control PCNA. For example, acetylation of lysine residues within the PIP-box weakens PARG interaction with PCNA in vitro. We aim to analyse quantitatively changes in acetylation and ubiquitination of PARG in response to DNA damage using a targeted proteomics approach. Using biochemical and biophysical assays, we will examine the importance of acetylated and ubiquitinated residues for PARG-PCNA interaction and modulation of binding parameters in response to DNA damage. Most importantly, we will investigate the importance of acetylated and ubiquitinated residues for the function of PARG in the context of DNA replication and DNA damage response. To this end, we will test how mutations in these residues affect PARG localization, mobility, recruitment to replication foci and DNA damage sites, and whether the mutations can rescue replication defects and DNA damage sensitivity of PARG-depleted cells. Overall, our results will further our insights on how the interaction of PARG with PCNA contributes to the regulation of the dynamic processes of DNA replication and repair. PCNA and PARG are of critical significance as clinical targets for cancer research. Whilst targeting a common interaction site on PCNA may cause general cytotoxicity, identification of specific residues within PARG that are regulated by acetylation and ubiquitination, and that are critical for PCNA interaction, should greatly facilitate targeted inhibitor design.

DNA replication and repair are essential cellular processes that ensure duplication of genomic material and safeguard the genome from deleterious mutations. Both processes need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair within replication factories. Poly(ADP-ribose) glycohydrolase (PARG) is an essential enzyme in eukaryotes that removes poly(ADP-ribose), interacts with PCNA and is required for replication and recovery from replication stress. In this project we analysed how the PARG-PCNA interaction is regulated in the context of replication fork dynamics and DNA damage response. We performed targeted mass spectrometry analysis of PARG acetylation sites in response to various types of DNA damage and replication stress, followed by functional characterization of their importance for PARG-PCNA interaction, PARG localization, mobility, recruitment to replication foci and DNA damage sites, replication stress response and DNA damage response. We found that stress-induced changes in PARG acetylation sites are not required for the regulation of dynamic interactions between PARG and PCNA. Instead, PARG-PCNA interaction through the PIP-box drives PARG-PCNA condensate formation in vitro and within replication factories in cells. We generated PARG HEK293 cells lacking the largest nuclear PARG isoform that show reduced proliferation and reduced fork rate, early S-phase arrest, impaired replication fork restart after replication stress exposure and sensitivity to HU and DNA-damaging agents, and thereby recapitulate replication phenotypes previously reported for shPARG-depleted cells. These phenotypes cannot be rescued with PARG PIP-box mutants, suggesting that PARG-PCNA interaction is required for the regulation of replication factories to ensure unperturbed replication and efficient response to replication stress. As part of this project, we also validated a newly established system, UV-FLIM-FRET, to monitor and quantify protein-protein interactions at DNA damage sites at a single cell level with high temporal resolution. Our study represents the first characterization of the effect of PCNA-interacting proteins on PCNA clustering through condensate formation and may put forth a new paradigm of how multivalent interactions dynamically modulate protein localization and abundance within replication factories.