Understanding the role of ADP-ribose chain length on PARP1/2 dynamics at sites of DNA damage and on the PARP inhibitor response

Thesis

Upon sensing DNA damage, PARP1/2 are recruited to lesions where they ADP-ribosylate themselves and other proteins at sites of damage, including histones, thus promoting, and directing DNA repair. Loss of catalytic activity of PARP1 causes its prolonged retention, or ‘trapping', at sites of DNA breaks, which can exacerbate existing damage. This is exploited therapeutically in cancers with deficiencies in DNA repair pathways, in which catalytic inhibition of PARP1 causes synthetic lethality. Recent years have seen an increase in studies investigating the mechanism of action of these drugs, PARP inhibitors, with sometimes contradicting findings.

HPF1, an essential PARP1/2 co-factor, discovered more than 50 years after PARP1, forms a complex with PARP1/2 at sites of DNA damage and switches ADP-ribosylation to target serine residues on histones and many other DNA damage associated proteins. While this function of HPF1 has been studied and shown to be important for DNA repair and to reduce PARP inhibitor sensitivity, HPF1 also restricts ADP-ribose chain elongation. This function has not been as extensively studied, and its role in regulating PARP1 release and DNA repair is yet not well understood. Therefore, I created cell lines that allow inducible expression of wild-type or catalytically impaired versions of both HPF1 and PARP1, to allow better study of the structure/function relationships of these proteins, including the investigation of the roles of mono- vs poly-ADP-ribosylation in the DNA damage response and cellular survival. Using these mutants, I demonstrate that mono-ADP-ribosylation, and not only poly-ADP-ribosylation, is important for the release of PARP1 from sites of DNA damage.

In this work, I also demonstrate that the release of catalytic PARP1 mutants is promoted by a novel PARP1-selective inhibitor, which adds to a growing field of research into how these drugs work. Finally, by using a catalytic PARP1 mutant, and this novel, PARP1-selective inhibitor, I am able to provide new insights into the relationship between PARP1 and PARP2 at sites of DNA damage.

Authors: Schützenhofer, K

• DOI: 10.5287/ora-yrvq6vozg
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: PARP inhibitor; replication stress; PARP trapping; ADP-ribosylation; DNA repair
• Subjects: Cancer; DNA repair; Molecular biology