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 (13)C, (15)N-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 dinucleotide biosynthesis, and cofactor recycling pathways with poly(ADP-ribose) polymerase-1 permitted labeling of PAR from (13)C-glucose and (13)C, (15)N-ATP in a single pot reaction. The scheme is efficient, yielding approximately 400 nmoles of purified PAR from 5 mumoles ATP, and the behavior of the synthetic PAR is similar to data from PAR synthesized by cell extracts. The resonances for (13)C, (15)N-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.
Schultheisz
Heather L
HL
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB33, La Jolla, California 92037, USA.
Szymczyna
Blair R
BR
Williamson
James R
JR
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D009536:Niacinamide / Q000737:chemistry / Q000378:metabolism; D009535:Nicotinamidase / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes; D009690:Nucleic Acid Conformation; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000737:chemistry*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009536:Niacinamide; D011064:Poly Adenosine Diphosphate Ribose; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D009535:Nicotinamidase; D005947:Glucose