Enzymatic Synthesis and Structural Characterization of 13C, 15N-Poly(ADP-ribose)

Poly(ADP-ribose) is a significant nucleic acid polymer involved with diverse functions in eukaryotic cells, yet no structural information is available. A method for the synthesis of C-13, N-15-poly(ADP-ribose) (PAR) has been developed to allow characterization of the polymer using multidimensional nuclear magnetic resonance (NMR) spectroscopy. Successful integration of pentose phosphate, nicotinamide adenine dinucieotide biosynthesis, and cofactor recycling pathways with poly(ADP-ribose) polymerase-1 permitted labeling of PAR from C-13-glucose and C-13, N-15-ATP in a single pot reaction. The scheme is efficient, yielding similar to 400 nmoles of purified PAR from 5 mu moles ATP, and the behavior of the synthetic PAR is similar to data from PAR synthesized by cell extracts. The resonances for C-13, N-15-PAR were unambiguously assigned, but the polymer appears to be devoid of inherent regular structure. PAR may form an ordered macromolecular structure when interacting with proteins, and due to the extensive involvement of PAR in cell function and disease, further studies of PAR structure will be required. The labeled PAR synthesis reported here will provide an essential tool for the future study of PAR-protein complexes.