Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation

Abstract

DNA-dependent poly(ADP-ribose) polymerases (PARPs) PARP1, PARP2 and PARP3 act as DNA break sensors signalling DNA damage. Upon detecting DNA damage, these PARPs use nicotine adenine dinucleotide as a substrate to synthesise a monomer or polymer of ADP-ribose (MAR or PAR, respectively) covalently attached to the acceptor residue of target proteins. Recently, it was demonstrated that PARP1-3 proteins can directly ADP-ribosylate DNA breaks by attaching MAR and PAR moieties to terminal phosphates. Nevertheless, little is still known about the mechanisms governing substrate recognition and specificity of PARP1, which accounts for most of cellular PARylation activity. Here, we characterised PARP1-mediated DNA PARylation of DNA duplexes containing various types of breaks at different positions. The 3′-terminal phosphate residue at doublestrand DNA break ends served as a major acceptor site for PARP1-catalysed PARylation depending on the orientation and distance between DNA strand breaks in a single DNA molecule. A preference for ADP-ribosylation of DNA molecules containing 3′-terminal phosphate over PARP1 auto-ADPribosylation was observed, and a model of DNA modification by PARP1 was proposed. Similar results were obtained with purified recombinant PARP1 and HeLa cell-free extracts. Thus, the biological effects of PARP-mediated ADP-ribosylation may strongly depend on the configuration of complex DNA strand breaks.One of the earliest DNA damage response events in the cell is the recruitment of DNA-dependent poly(ADP-ribose) polymerases 1, 2 and 3 (PARP1-3) to the sites of DNA strand breaks 1-3 . PARPs 1-3 are catalytically activated through interaction with DNA strand discontinuities and catalyse poly(ADP-ribosyl)ation (PARylation) or mono(ADP-ribosyl)ation (MARylation, in case of PARP3) of nuclear acceptor proteins including auto-ADP-ribosylation using NAD + as the ADP-ribose donor 4-6 . Protein ADP-ribosylation provides a scaffold for the recruitment of other proteins, which also become potential targets for PARP-dependent ADP-ribosylation altering the function of the modified proteins and coordinating the choice of DNA break processing and repair pathways. PARP1 is one of the most abundant nuclear proteins and accounts for ~80-90% of the PARylation activity in the cell induced by DNA damage 7,8 . PARP1 is recruited to damage sites in genomic DNA within a few seconds after laser micro-irradiation 3 and modulates multiple pathways involved in DNA strand break repair: base excision repair, nucleotide excision repair, homologous recombination and non-homologous end-joining 4,9 . Depletion of PARP1 results in hypersensitivity to ionising radiation, to oxidative stress, and to alkylating agents 10 .Recently, the previously unknown phenomenon of post-replicative reversible ADP-ribosylation of DNA strand break termini catalysed by mammalian PARP1-3 was uncovered. These PARPs catalyse covalent addition of ADP-ribose units to 5′-and 3′-terminal phosphates and to 2′-OH termini of modified nucleotides at DNA strand breaks, thereb...

NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential

“…The DNAbound PARPs, such as PARP1-3, can attach the mono-ADP-ribose (MAR) or poly-ADP-ribose (PAR) moieties directly to the DNA breaks. [153][154][155][156] Meanwhile, PARPs also catalyze the ADPribosylation of various proteins that facilitate the chromatin relaxation and the recruitment of repair factors. [157][158][159][160][161][162] The effect of PARPs to stimulate chromatin decompaction might be exerted via the steric hindrance of PAR chain, the negative charge of DNA and PAR, or the displacement of core histones.…”

Progress and outlook in studying the substrate specificities of PARPs and related enzymes

“…Accordingly, ADP-ribosylation may turn out to be a post-replicative DNA modification and a post-transcriptional RNA modification in addition to being a protein PTM. PARP1, PARP2 and PARP3 have been shown to catalyse DNA ADP-ribosylation [66][67][68][69][70], while PARP10, PARP11 and PARP15 modify RNA, producing what can be described as a noncanonical RNA cap [71]. RNA is also efficiently ADP-ribosylated in vitro by TRPT1/Tpt1 [71,72], a highly diverged PARP-like protein that is sometimes referred to as the 18th member of the PARP/ARTD superfamily [8].…”

“…RNA is also efficiently ADP-ribosylated in vitro by TRPT1/Tpt1 [71,72], a highly diverged PARP-like protein that is sometimes referred to as the 18th member of the PARP/ARTD superfamily [8]. Although studies that demonstrate these activities were prevalently conducted in vitro, both the concentration of enzymes and of NAD + , which are compatible with physiological values, as well as the features of nucleic acid substrates used in biochemical assays, strongly suggest that the same reactions may occur in cells [66][67][68][69][70]. Experiments with cell-free extracts and preliminary in vivo observations also point in that direction [68][69][70].…”

“…Although studies that demonstrate these activities were prevalently conducted in vitro, both the concentration of enzymes and of NAD + , which are compatible with physiological values, as well as the features of nucleic acid substrates used in biochemical assays, strongly suggest that the same reactions may occur in cells [66][67][68][69][70]. Experiments with cell-free extracts and preliminary in vivo observations also point in that direction [68][69][70]. Finally, ADP-ribosylation of both DNA and RNA can be reversed in vitro by multiple ADP-ribosylglycohydrolases, including canonical (i.e.…”

“…Finally, ADP-ribosylation of both DNA and RNA can be reversed in vitro by multiple ADP-ribosylglycohydrolases, including canonical (i.e. macrodomain-containing) and noncanonical erasers [11,[66][67][68][69][70]73].…”