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

Elie Matta,Bakhyt T Matkarimov,Alexander A Ishchenko,Bekbolat Khassenov,Assel Kiribayeva

doi:10.1038/s41598-020-60631-0

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 double-strand 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-ADP-ribosylation 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.

Highlights

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[1,2,3]
It has been demonstrated elsewhere that PARP2- and PARP3-catalysed DNA ADP-ribosylation is strongly dependent on the distance between breaks in DNA substrates[12]
For optimisation of poly(ADP-ribose) polymerase 1 (PARP1) DNA PARylation activity we performed an in vitro assay at a saturating concentration of NAD+ (1 mM) with the human PARP1 enzyme and various 32P-radiolabelled Dbait-based DNA structures (Supplementary Table S1) containing a one-nucleotide gap for PARP1 activation and 5′- or 3′-terminal phosphates as acceptor groups of various overhangs at a unique DSB end

Summary

Introduction

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[1,2,3]. 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, thereby producing a covalent MAR–DNA or PAR–DNA adduct[11,12,13]. This discovery provides novel molecular insights into PARPs’ functions. Possible functional interactions between PARP1-mediated PARylation and formation of 3′-phosphorylated breaks are discussed

Methods

Results

Conclusion