DNA Strand Break-Mediated Partitioning of Poly(ADP-Ribose) Polymerase Function

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Abstract
The nuclear enzyme poly(ADP-ribose) polymerase participates in DNA excision repair. Following binding to DNA strand breaks through its amino-terminal Zn(2+)-finger domain, the enzyme is activated to form polymerase-associated ADP-ribose polymers of various sizes. Focusing on this "automodification" reaction, we observed that optimal enzyme activity and maximal polymer formation were attained only at a strict stoichiometry of two polymerase molecules per DNA fragment. Using various linearized DNAs and nicked circular DNA, we show that this stoichiometric dependence is dictated by the number of enzyme activating sites, i.e., DNA strand breaks. Deviations from the optimal ratio inevitably resulted in decreased polymer formation, ruling out a strict automodification mechanism of poly(ADP-ribosyl)ation. Our results suggest that the mechanism of poly(ADP-ribose) formation on polymerase molecules entails DNA strand break-mediated partitioning of the polymerase into two functional populations: one bound to the DNA breaks and catalytically active, the other, catalytically inactive, functioning as polymer acceptors.

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