Proteolysis of poly(ADP-ribose) polymerase by caspase 3: kinetics of cleavage of mono(ADP-ribosyl)ated and DNA-bound substrates.
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents and during DNA base excision repair. After DNA damage, the enzyme binds to nicks in DNA through its N-terminal zinc fingers and catalyzes the formation of poly(ADP-ribose) on various nuclear acceptors including itself. When DNA damage is extensive, cells induce their own demise by activating the proteases that induce apoptosis (caspases) which cleave PARP and other death substrates. Here we report the development of a new approach to investigate the sensitivity of mono(ADP-ribosyl)ated and DNA-bound PARP to cleavage during apoptosis. The development of a stoichiometric labeling procedure of the enzyme has allowed us to evaluate the catalytic properties of caspase 3 toward mono(ADP-ribosyl)ated PARP at various enzyme:substrate molar ratios. We show that low levels of automodification (< or = 3 U of ADP-ribose per chain) do not inhibit the proteolysis of the substrate. In addition, we demonstrate that binding of unmodified PARP to DNA influences the kinetics of its cleavage by caspase 3.
D'Amours
D
D
Poly(ADP-ribose) Metabolism Group, Unit of Health and Environment, CHUL Research Center and Laval University, Ste-Foy, Quebec, Canada.
Germain
M
M
Orth
K
K
Dixit
V M
VM
Poirier
G G
GG
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3; D020169:Caspases; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D007700:Kinetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
D000246:Adenosine Diphosphate Ribose; D004247:DNA; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases