Molecular mechanism of poly(ADP-ribosyl)ation catalyzed by human poly(ADP-ribose) polymerase-1
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Human poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear enzyme which catalyzes protein poly(ADP-ribosyl)ation upon binding to DNA. NAD+ is used as a co-substrate in the reaction via iterative transfer of its ADP-ribose moiety to acceptor proteins including PARP-1 itself, yielding elongated and branched poly(ADP-ribose) (PAR) polymers. This type of protein posttranslational modification has been demonstrated in the regulation of diverse biological processes including DNA repair, gene expression, cell cycle, etc. Therefore, elucidating the catalytic mechanism of PARP-1 would not only advance our understanding of how its enzymatic activity is regulated under physiological and pathophysiological conditions, but also greatly benefit the development of novel therapeutics involving pharmacological manipulation of PARP-1. In this dissertation, the molecular mechanism of DNA-dependent poly(ADP-ribosyl)ation by human PARP-1 was addressed from an enzymological perspective in terms of the allosteric ligand DNA, the substrate NAD+, and the PARP-1 protein–DNA complex as a whole. By site-specific labeling of the DNA-binding domain AB of PARP-1 and DNA ligands with fluorophores, quantitative binding kinetics of AB with DNA was investigated by single-molecule fluorescence spectroscopy. Two binding modes, one involving a strongly-associated protein–DNA complex and the other being transient, were suggested by the experimental data. To probe the catalytic mechanism of the initiation, elongation, and branching step of poly(ADP-ribosyl)ation with regard to NAD+ substrate scope, analogues of NAD+ with fluoro-substituted ribose ring were synthesized chemoenzymatically and employed as substrates. The results are consistent with the proposed mechanism that the ADP-ribosyl transfer reaction proceeds through an oxocarbenium-like transition state. Mass spectrometry and biochemical approaches were utilized to decipher the poly(ADP-ribosyl)ation sites on PARP-1 and the chemical nature of PAR–protein linkages. The data confirm the existence of automodification sites beyond domain D, and lysine could be the targeted residue for poly(ADP-ribosyl)ation, either enzymatically or nonenzymatically. The macromolecular mechanism of DNA-dependent PARP-1 automodification was established by an in vitro radioactivity-based poly(ADP-ribosyl)ation assay using structurally distinguishable PARP-1 mutants. The data support the model of an intermolecular process. Top-down MS analysis and crosslinking assay bolster the monomeric structure of domain C in solution and its participation in interdomain contacts during PARP-1 catalysis. Taken together, these mechanistic studies provide further insight into the catalytic strategies exploited by human PARP-1 complementary to recent reports of structural characterization, and may help discover better therapeutic agents modulating poly(ADP-ribosyl)ation.