DNA integrity is constantly challenged by genotoxic stress. These threatening stimuli can arise from exogenous sources (IR, UV, chemicals) but also be generated within the cellular environment, such as during replication and transcription. Double strand breaks (DSBs), in which both DNA strands are severed, are a common type of toxic intermediate that can be generated upon genotoxic insults. DSBs can be highly mutagenic, as they can trigger chromosome rearrangements that ultimately can induce tumorigenesis or extensive cell death. Cells are equipped with several DNA repair systems that in turn detect damages and elicit DNA repair through the sophisticated action of multiple pathways that ultimately lead to the preservation of genomic stability. However, during gametogenesis DSBs are deliberately induced in order to promote inter-homologue recombination and ultimately establish chiasmata (the cytological manifestation of an occurred crossover), which are essential for faithful chromosome segregation. Therefore, in the germ cells these repair systems have to deal with both unscheduled and physiologically induced DSBs.
Poly(ADP-ribosyl)ation (PARylation) is an extremely transient post-translational modification that involves the addition of poly(ADP-ribose) moieties (PAR) on target proteins, synthetized using NAD+ as a substrate. Parp1 and Parp2 (PARPs), the two main PAR polymerases, exert a pivotal role in DNA damage response, as they directly target DNA for repair or promote the recruitment of repair factors to the chromatin. Indeed, mammalian Parp1-/- and Parp2-/- mutant cells are viable but display hypersensitivity to DNA damage, whereas the Parp1-/- Parp2-/- double knockout is embryonic lethal, suggesting a partial functional redundancy. PAR is promptly removed from the target proteins by Poly(ADP-ribose) glychohydrolase PARG, which is an essential protein.
The lethality displayed by Parp1/2 double and PARG single mutants has prevented the study of PARylation dynamics in a null background in vivo, and most of the knowledge gathered so far has been relying on the use of RNAi (which can give rise to partial phenotypes) or chemical inhibitors (which can produce “off target” effects).
I am interested in studying the requirements of Poly(ADP-ribosyl)ation in the DNA damage response in vivo, and specifically in the germline, by using Caenorhabditis elegans as a model system. Specifically, the aim of my proposed research is to clarify the functions of PARPs and PARG under physiological conditions of growth and upon different types of genotoxic insults, known to elicit the response of different repair pathways, in order to identify the specific DNA repair ensembles that require PARylation. Ultimately, my project also aims at the identification of novel PARPs and PARG interactors and PARylated substrates by using a proteomic approach.