DNA recognition and mechanistic investigation of poly(ADP-ribose) polymerase-1

Human PARP-1 is a nuclear protein containing six functional domains that catalyzes the poly(ADP-ribosyl)ation of a variety of protein substrates including itself. This process involves consumption of NAD+ as the ADP-ribose donor for forming the poly(ADP-ribose) (i.e., PAR). PARP-1 utilizes this polymerization reaction to effect its regulatory role in many important biological processes including transcription and DNA repair. Activation of PARP-1 self-modification requires the presence of damaged DNA. Activity levels of PARP-1 differ depending on the type of DNA lesion. It is proposed that the differences in activity level of PARP-1 are related to the binding stoichiometry of PARP-1 to DNA. Using double and single stranded break DNA mimics, stoichiometric analyses of PARP-1 were performed using sedimentation velocity techniques. In both cases, PARP-1 forms both 1:1 and 2:1 protein:DNA complexes, consistent with protein dimer formation. Correlation of PARP-1 activity with the DNA structure it encounters can also be explained by the utilization of different functional domains for DNA recognition. To investigate this hypothesis, DNA-binding domain AB was labeled with the Cy3 fluorophore, and a Cy5-labeled DNA with a double stranded break, was used as its interaction counterpart. Protein-DNA interactions were monitored by single molecule fluorescence colocalization. It was observed that a 2:1 protein:DNA complex is formed. Furthermore, recognition of double stranded DNA breaks by domain AB involves two different binding steps with distinct dissociation kinetics. Finally, two FRET states were observed as domain AB interacted with DNA. This suggests that domain AB utilizes different regions to interact with DNA during the recognition process. After activation, PARP-1 synthesizes poly(ADP-ribose) through three catalytic processes: initiation, elongation and branching. To study the domain requirements for polymer synthesis, truncated PARP-1 constructs ABC and DEF were tested. It was observed that initiation and elongation of short polymers requires the presence of DEF, ABC and DNA. As the polymer gets larger, DEF itself is capable of adding ADP-ribose onto the PAR polymers. Current data is consistent with a mechanistic proposal where automodification of PARP-1 happens intramolecularly and PAR elongation takes place at the distal end of the growing polymer.