Study of DNA ADP-ribosylation and Its Role in DNA Damage Response – DNAPAR18

Cellular DNA is constantly damaged by exogenous and endogenous factors resulting in base damage and DNA strand breaks. The accumulation of endogenously occurring DNA lesions is a primary factor contributing to aging. Failure to detect and repair DNA strand breaks and other lesions can lead to deleterious consequences for the cell such as chromosomal aberrations, genomic instability and cell death. Poly(ADP-ribose) polymerases (PARPs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARP1-3 act as DNA break sensors, which regulate the DNA repair pathways by recruiting chromatin remodeling and DNA repair factors to DNA breaks and can promote cell death at a high level of DNA damage. Our recent in vitro studies showed for the first time that mammalian PARP1 and PARP2 can catalyse covalent addition of ADP-ribose units to terminal phosphates and to 2’-OH termini of modified nucleotides at DNA strand breaks, producing a covalent PAR–DNA adduct. Moreover, we demonstrated that PARP3 can effectively produce mono(ADP-ribose)-DNA (MAR-DNA) adducts on terminal phosphate residues at DSB and SSB termini, sharing its substrate specificity with PARP2 and revealing a DNA break-oriented mechanism of DNA ADP-ribosylation by PARP3 or PARP2. We found that depending on configuration of DNA strand breaks, the DNA termini can become preferred acceptor sites for ADP-ribosylation as compared to proteins. We demonstrated that ADP-ribosylated gapped DNA could be ligated to yield continuous dsDNA containing an aberrant abasic site. Our findings reveal effective DNA PARylation activity in cell-free extracts and provide indirect evidence of the presence of PAR–DNA adducts in purified genomic DNA samples after genotoxic treatment. These results suggest that certain types of complex DNA breaks can be effectively ADP-ribosylated by PARPs in cellular response to DNA damage. Importantly, DNA ADP-ribosylation is a reversible process since PAR can be entirely degraded by Poly(ADP-ribose) glycohydrolase (PARG). This discovery provide novel molecular insights into the PARPs function. ADP-ribosylation of DNA strand break termini may: temporary block their processing and protect them from non-specific degradation or aberrant error-prone end joining; enable an apoptotic signal if not removed; trigger relocation of the damaged DNA loci within the nucleus or stable PAR/MAR-guided recruitment and assembly of the repair factors. A major issue is now to provide definitive evidence of DNA ADP-ribosylation in live cells and characterize its physiological relevance
The major aim of the present project is to reveal the role of DNA ADP-ribosylation in DNA damage response and in coordination of DNA strand break repair with unique new tools of high sensitivity and specificity that will be set up during this project. Here, we propose: (i) to further characterize DNA substrate specificity and mechanisms of DNA PARylation by PARPs; (ii) to address the role of DNA ADP-ribosylation in non-homologous end joining; (iii) to elaborate advanced mass spectrometry analysis of ADP-ribosylated nucleotides; (iv) to produce antibodies specific for ADP-ribosylated-DNA adducts; (v) identify the ADP-ribosylated DNA adducts and their potential “readers” or “erasers” in mammalian cells exposed to various genotoxic treatments. Completion of these tasks will provide high-value research tools for identification of ADP-ribosylated DNA adducts and will enable us to identify their potential biological functions. Finally, the new knowledge about the mechanisms of PARPs actions will help to identify novel therapeutic or diagnostic targets in diseases associated with age, inflammation and DNA damage.