Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

• Published in: DNA repair 2008-11, Vol.7 (11), p.1787-1798
• Main Authors: Kedar, Padmini S., Stefanick, Donna F., Horton, Julie K., Wilson, Samuel H.
• Format: Article
• Language: English
• Subjects: Animals; Ataxia Telangiectasia Mutated Proteins; ATR; Bacteriology; Biological and medical sciences; Cell Cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - physiology; DNA Methylation; Enzyme Inhibitors - pharmacology; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Growth, nutrition, cell differenciation; Humans; Immunoprecipitation; Methylation damage; Mice; Microbiology; Models, Biological; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; PARP inhibitor; PARP-1; Poly(ADP-ribose) Polymerases - metabolism; Protein Binding; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - physiology; Recombinant Proteins - chemistry; S Phase; Signal Transduction
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2008.07.006

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.