Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition.
Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.
Kedar
Padmini S
PS
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Stefanick
Donna F
DF
Horton
Julie K
JK
Wilson
Samuel H
SH
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008954:Models, Biological; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D016196:S Phase; D015398:Signal Transduction
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; C576897:Atr protein, mouse; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases