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Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase
H Mendoza-Alvarez1, R Alvarez-Gonzalez
1Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
Abstract:
Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.
Insights
This study biochemically characterizes mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP). Findings show PARP modification occurs exclusively as single ADP-ribose units, primarily within its automodification domain, not polymeric chains.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Poly(ADP-ribose) polymerase (PARP) is a key enzyme in DNA repair.
- Understanding PARP's catalytic mechanism, particularly its ADP-ribosylation activity, is crucial.
Purpose of the Study:
- To biochemically characterize the mono(ADP-ribosyl)ation of PARP.
- To identify the specific sites and products of PARP ADP-ribosylation.
Main Methods:
- Activity gel assays and SDS-PAGE with [32P]-3'-dNAD+.
- Chemical release of enzyme-bound ADP-ribose units.
- High-resolution polyacrylamide gel electrophoresis and peptide mapping.
Main Results:
- PARP undergoes mono(ADP-ribosyl)ation with [32P]-3'-dNAD+ at low substrate concentrations.
- Only mono(ADP-ribosyl)ated products (3'-dAMP) were observed, not polymers.
- ADP-ribosylation occurred on up to four residues within a 22 kDa automodification domain.
Conclusions:
- PARP initiates ADP-ribosylation by adding single 3'-dADP-ribose units.
- The automodification domain is the primary site for PARP mono(ADP-ribosyl)ation.
- The initiation of ADP-ribosylation by PARP is significantly slower than polymerization.