Defining the role of PARPs in the DNA repair and genome stability

Preserving the integrity of genetic material through repair of damaged DNA is critical for the health of an organism. DNA repair mechanisms that resolve different DNA lesions are becoming increasingly well defined. However, a remaining challenge is to understand how these processes integrate to allow cell survival if a particular repair pathway fails. Deciphering these complex interactions will define the mechanistic basis of genome stability and uncover strategies to manipulate these pathways for clinical benefit. My laboratory is focussed on addressing these important questions, with specific reference to how a set of enzymes called Poly(ADP-ribose)-polymerases (PARPs) regulate DNA repair.

The best defined role of PARPs is in promoting repair of breaks in the DNA double strand helix. Inhibition of this pathway using small molecule PARP inhibitors (PARPi) is being exploited in the clinic to kill tumours with defects in a DNA repair process known as homologous recombination (HR). Our current understanding of this process is that PARPi can 'trap' two enzymes, PARP1 and PARP2 at DNA breaks. These PARP-DNA adducts then blocks enzymes that copy genetic material during DNA replication, causing DNA damage that requires HR for repair. Therefore, in the absence of HR, cells are killed due to their inability to repair PARPi-induced replication blocks. Importantly, our previous MRC funded work indicated that there is significant functional overlap in these mechanisms and that PARP1/PARP2 not only promote repair of DNA breaks, but also resolution of replication blocks. The proposed work will build on these key conceptual advances in addition to exciting new data from my laboratory to further define how PARPs promote genome integrity. Specifically, we will characterise an additional PARP-dependent mechanism to repair replication blocks, in addition to a novel gene that can compensate for loss of PARP1/PARP2.

These studies will not only increase our understanding of fundamental principles that allow cells to maintain genome integrity, but also provide critical information that will underpin development more specific PARPi with increased efficacy in the clinic. Moreover, identifying genes that are critical for cell viability when PARPs are disrupted will identify strategies to broaden the use of these agents to treat tumour types with defects in pathways other than HR.

This work will increase our understanding of how Poly(ADP-ribose)-polymerases (PARPs) maintain genome integrity through replication repair and characterise pathways that can compensate for loss of these processes. We have exploited genome editing to generate cell lines defective in the principle DNA damage responsive PARPs alone, or in combination. Using this unique set of reagents in combination with siRNA depletion of DNA repair factors, we have identified PARP1/PARP2 regulate HR-dependent and -independent mechanisms for repair of stalled/damaged replication forks. Moreover, through a genome-wide CRISPR/Cas9 screen designed to identify genes that are synthetic lethal with parp1/parp2 gene disruption, we discovered a novel gene that is required for cells to tolerate replication stress.

Building on this work, we will define the mechanistic basis of these pathways. Using genome editing technology, in combination with cell based assays to monitor replication dynamics, DNA damage response activation, cell cycle progression and genome stability, we will define how PARP1/PARP2 regulate HR-dependent and independent DNA repair. Using cutting edge mass spectrometry, we will map ADP-ribosylation sites targeted during replication stress and through mutational analysis define the mechanistic basis of how these modifications regulate genome stability. Additionally, we will assess the role of a novel DDR gene that can compensate for loss of PARP1/PARP2-dependent replication repair mechanisms, identifying the pathway it regulates and the molecular basis of its function. Together these experiments will provide important information regarding how PARP1/PARP2 regulate replication-associated repair and the pathways that can compensate for loss of these processes. This will inform strategies to improve the efficacy of PARP inhibitors in the clinic and identify new synthetic lethal interactions to broaden the use of these agents to treat other tumour types.