Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis
Damien D’Amours1,
Frédéric R. Sallmann2,
Vishva M. Dixit3 and
Guy G. Poirier4,*
1 Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, UK
2 Department of Biology, MIT 77, Massachusetts Avenue, Cambridge, MA, USA
3 Department of Molecular Oncology, Genentech Inc., 460 Point San Bruno Blvd, South San Francisco, CA 94080, USA
4 Unit of Health and Environment, Laval University Medical Research Center, CHUQ, Faculty of Medicine, Laval University, Québec, Canada
View larger version (45K):
[in a new window]
|
Fig. 2. Affinity purification of the apoptotic fragments of PARP-1. Bovine PARP-1 (purified to homogeneity) was cleaved with caspase 3 and subjected to ssDNA-cellulose affinity chromatography as described in Material and Methods. Silver-stained SDS-polyacrylamide gel (Desnoyers et al., 1995) showing the apoptotic fragments of PARP-1 obtained in late ( 500 mM NaCl; 24 kDa) and early (89 kDa) fractions of the chromatography procedure.
|
|
View larger version (16K):
[in a new window]
|
Fig. 3. Biochemical properties of the N-terminal apoptotic fragment of PARP-1. The catalytic activity of native PARP-1 was determined in the presence of increasing amounts of the 24 kDa apoptotic fragment of PARP-1. The results are expressed as percentage of maximal activity obtained in the absence of apoptotic fragment. Experiments were performed in triplicate at subsaturating concentrations of DNA strand-breaks, as described in Materials and Methods.
|
|
View larger version (29K):
[in a new window]
|
Fig. 4. Transdominant inhibition of DNA base excision repair by the N-terminal apoptotic fragment of PARP-1. DNA BER was assayed by monitoring the repair of oxy-radical damaged plasmids in whole cell extracts from the normal lymphoblastoid cell line GM01953C. (A) Agarose gel showing the repair of nicked plasmids (OC) following incubation in whole cell extract containing growing concentrations of the N-terminal apoptotic fragment of PARP-1. (B) Densitometric integration of the gel shown in (A). Repair is expressed as the proportion of repaired plasmids (CC) over the total amount of plasmids used in the repair reaction (OC+CC). Experiments were performed in the presence of a saturating concentration of NAD+ (2 mM).
|
|
View larger version (24K):
[in a new window]
|
Fig. 5. Inhibition of poly(ADP-ribose) synthesis in cells by overexpression of the apoptotic (24 kDa) DBD of PARP-1. (A) Immunoblotting of the overexpressed DBD of PARP-1. Cultured cells were incubated for 16 hours with 1 mM dexamethasone to induce the expression of the DBD, and then extracted in SDS-PAGE loading buffer. 100,000 cells were loaded per well and analyzed by western blotting and immnodetection with the monoclonal antibody (F1-23), which recognizes the second zinc finger of PARP-1. (B) Transdominant inhibition of poly(ADP-ribose) synthesis by the apoptotic DBD of PARP-1 in cells treated with MNNG for 30 minutes. Different concentrations of MNNG were used as indicated on the histogram. The inhibition of pADPr synthesis is expressed as the % fraction of polymers detected in the presence of the 24 kDa DBD (+dexamethasone) over that detected in the absence of the fragment (-dexamethasone). The polymer levels detected in the absence of dexamethasone induction represent 100% PARP activity and are as follows: 185±3.7 units, 0 µM MMNG; 215±8.7 units, 20 µM MMNG; 430±17.3 units, 40 µM MMNG; and 837±15.6 units, 60 µM MMNG. Polymer levels were measured by quantitative immunodot blot (Affar et al., 1998) and are expressed as arbitrary fluorescence units per 4x106 cells. The results plus s.d. of three independent experiments are shown.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2001
) and proteolysed (
) PARP-1 were compared for their poly(ADP-ribosyl)ation activity in the presence of saturating concentration of co-enzymic DNA over a period of 5 minutes. The activity of native PARP-1 was also measured in the absence of DNA (
). Poly(ADP-ribose) synthesis was carried out until maximal level of product could be obtained for both the cleaved and intact enzyme in order to maximize the potential re-association of the apoptotic fragments. This approach was successfully used for the reconstitution of PARP-1 activity following limited proteolysis with papain (