Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.
Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.
Cancer Research Laboratory, Québec Genomic Center, Laval University - CHUQ Research Center, Québec, Canada.
Jean-Philippe
JP
Gagné
Emilie
E
Pic
Maxim
M
Isabelle
Jana
J
Krietsch
Chantal
C
Ethier
Eric
E
Paquet
Isabelle
I
Kelly
Michel
M
Boutin
Kyung-Mee
KM
Moon
Leonard J
LJ
Foster
Guy G
GG
Poirier
D004249:DNA Damage; D004260:DNA Repair; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D046912:Multiprotein Complexes / Q000302:isolation & purification; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D060066:Protein Interaction Maps; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013312:Stress, Physiological / Q000235:genetics
D046912:Multiprotein Complexes; D020543:Proteome; D011065:Poly(ADP-ribose) Polymerases