The Arabidopsis thaliana Poly(ADP-Ribose) Polymerases 1 and 2 Modify DNA by ADP-Ribosylating Terminal Phosphate Residues.

Sabira Taipakova,Alexander A Ishchenko,Didier Gasparutto,Murat Saparbaev,Regina Groisman,Aigerim Kuanbay,Yeldar Baiken,Amangeldy K Bissenbaev,Christine Saint-Pierre

doi:10.3389/fcell.2020.606596

Abstract

Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyze the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are viewed as DNA damage sensors that, upon binding to strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. The flowering plant Arabidopsis thaliana contains three genes encoding homologs of mammalian PARPs: atPARP1, atPARP2, and atPARP3. Both atPARP1 and atPARP2 contain poly(ADP-ribosyl)ating activity; however, it is unknown whether they could covalently modify DNA by ADP-ribosylating the strand break termini. Here, we report that similar to their mammalian counterparts, the plant atPARP1 and atPARP2 proteins ADP-ribosylate 5′-terminal phosphate residues in duplex DNA oligonucleotides and plasmid containing at least two closely spaced DNA strand breaks. AtPARP1 preferentially catalyzes covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 5′-phosphate, whereas atPARP2 preferentially ADP-ribosylates the nicked and gapped DNA duplexes containing the terminal 5′-phosphate. Similar to their mammalian counterparts, the plant PARP-catalyzed DNA ADP-ribosylation is particularly sensitive to the distance that separates two strand breaks in the same DNA molecule, 1.5 and 1 or 2 turns of helix for atPARP1 and atPARP2, respectively. PAR glycohydrolase (PARG) restored native DNA structure by hydrolyzing the PAR–DNA adducts generated by atPARPs. Biochemical and mass spectrometry analyses of the PAR–DNA adducts showed that atPARPs utilize phosphorylated DNA termini as an alternative to protein acceptor residues to catalyze PAR chain synthesis via phosphodiester bond formation between C1′ of ADP-ribose and a phosphate residue of the terminal nucleotide in DNA fragment. Taken together, these data establish the presence of a new type of DNA-modifying activity in Arabidopsis PARPs, suggesting a possible role of DNA ADP-ribosylation in DNA damage signaling and repair of terrestrial plants.

Highlights

Land plants are under constant exposition to a variety of abiotic stresses including UV radiation, droughts, temperature variation, salinity, and other environmental extremes that can extensively damage cellular DNA
We have demonstrated that in vitro mammalian PARP1 and PARP2 proteins can poly(ADPribosyl)ate duplex oligonucleotides containing multiple closely spaced DNA strand breaks and phosphorylated termini
We examined the biochemical activities of the purified atPARP1 and atPARP2 proteins using DNA substrates containing more than two strand breaks to mimic clustered DNA damage and repair intermediates: ExoARexTNick and ExoARexTgap, which are 40-mer oligonucleotide duplexes containing a nick and onenucleotide gap, respectively, composed of a 40-mer (RexT, referred to as S1) template strand and two complementary strands: 21-mer (ExoA) and phosphorylated 18-mer (5 pExo18) or 19-mer (5 pExo19) strands (Supplementary Table S1)

Summary

Introduction

Land plants are under constant exposition to a variety of abiotic stresses including UV radiation, droughts, temperature variation, salinity, and other environmental extremes that can extensively damage cellular DNA. Oxidative damage to DNA caused by ROS is believed to be a major source of genome instability and aging (Cadet and Wagner, 2013). It is agreed that PARP1, PARP2, and PARP3 are sensors of DNA damage that are activated by binding to DNA strand discontinuities. After activation of the catalytic domain, PARPs poly/monoADP-ribosylate (PARylate/MARylate) themselves and different nuclear proteins, these in turn regulate the functions of ADPribosylated proteins. It should be stressed that the PARP-catalyzed covalent protein posttranslational modification is a reversible process since PAR is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) which hydrolyze the ribose– ribose bonds in the polymer. The activation of PARPcatalyzed auto-ADP-ribosylation and modification of nuclear proteins such as histones is one of the common cellular responses to DNA damage (De Murcia and Menissier De Murcia, 1994)

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Conclusion