Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

  • Nucleic Acids Res. 2016 Nov 2;44(19):9279-9295. doi: 10.1093/nar/gkw675.
Ibtissam Talhaoui  1  2 ,  Natalia A Lebedeva  3 ,  Gabriella Zarkovic  1  2 ,  Christine Saint-Pierre  4 ,  Mikhail M Kutuzov  3 ,  Maria V Sukhanova  3 ,  Bakhyt T Matkarimov  5 ,  Didier Gasparutto  4 ,  Murat K Saparbaev  1  2 ,  Olga I Lavrik  6  7 ,  Alexander A Ishchenko  8  2
Affiliations
  • 1. Laboratoire «Stabilité Génétique et Oncogenèse» CNRS, UMR 8200, Univ. Paris-Sud, Université Paris-Saclay, F-94805 Villejuif, France.
  • 2. Gustave Roussy, Université Paris-Saclay, F-94805 Villejuif, France.
  • 3. SB RAS Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av. 8, Novosibirsk 630090, Russia.
  • 4. Université Grenoble Alpes, CEA, INAC/SPrAM UMR5819 CEA CNRS UGA, F-38000 Grenoble, France.
  • 5. National Laboratory Astana, Nazarbayev University, Astana 010000, Kazakhstan.
  • 6. SB RAS Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av. 8, Novosibirsk 630090, Russia [email protected].
  • 7. Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia.
  • 8. Laboratoire «Stabilité Génétique et Oncogenèse» CNRS, UMR 8200, Univ. Paris-Sud, Université Paris-Saclay, F-94805 Villejuif, France [email protected].
PMID: 27471034 DOI: 10.1093/nar/gkw675
Abstract

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3'-cordycepin, 5'- and 3'-phosphate and also to 5'-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5'-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2'-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2',1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1' of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.