Direction of elongation of poly(ADP-ribose) chains. Addition of residues at the polymerase-proximal terminus.

The mechanism of elongation of poly(ADP-ribose) on poly(ADP-ribose) polymerase was examined in two ways. The first technique involved a pulse-chase protocol. Poly(ADP-ribose) polymerase was labeled with radioactive NAD, excess precursor was removed by rapid gel filtration chromatography, and nonradioactive NAD was supplied for a second incubation. The products were released with alkali and digested with venom phosphodiesterase which generates AMP uniquely from the distal terminus. The distal residue that was labeled during the pulse remained at the distal terminus and was not converted to an internal residue during the chase. The second technique employed the NAD analog, 2'-deoxyNAD (dNAD), which can engage in mono-ADP-ribose addition reactions but lacks the 2'-OH that is required for polymer formation. dNAD inhibits ADP-ribose incorporation competitively but is not incorporated at the enzyme-distal chain terminus. These findings are inconsistent with a model of poly(ADP-ribose) synthesis in which new residues are added to the 2'-OH terminus of the growing chain, distal to the polymerase attachment. They are consistent with the alternative possibility that new residues are added at the 1" terminus, adjacent to the polymerase. Any such "proximal addition" model requires that there be at least two active center sites (akin to the ribosomal A and P sites), which at a certain stage of each elongation cycle will be occupied by ADP-ribose monomers and ADP-ribose polymers, respectively. Although dNAD does not enter poly(ADP-ribose), it does engage in a slow side reaction whereby a single dADP-ribose residue is added covalently to the polymerase itself, thereby inactivating the enzyme.