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Poly(ADP-ribose) polymerase 1 (PARP1) is an important first responder in the mechanism of DNA repair in eukaryotic cells. It is also a validated drug target, with four different PARP inhibitors (PARPi) approved for the treatment of BRCA-negative cancers. Despite past efforts, many aspects of PARPi are poorly understood, in particular their ability to trap PARP1 on chromatin and the relationships between their potencies, cellular toxicities, and trapping efficiencies. Because PARP trapping is widely believed to originate in allosteric coupling between DNA binding and the catalytic site, we further investigated the binding properties of PARP1 to a model for DNA with a double-strand break in the presence and absence of PARPi. Specifically, we have used sequential mixing stopped-flow spectroscopy to identify a slow conformational change that follows rapid DNA binding. Using a range of DNA concentrations and different mutants of PARP1 we demonstrate that this conformational change is one of the steps of the "monkey bar mechanism" that promotes DNA-dependent dissociation of DNA. This conformational change also corresponds to the previously identified conformational change associated with DNA-dependent activation of PARP1. Despite linking the conformational change associated with DNA binding and release to DNA activation, we find no evidence for PARPi perturbing this allosteric coupling.

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: D(eff), the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. D(eff) for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in the response to DNA damage and is the target of four different clinical inhibitors used for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation via destabilization of a subdomain of the catalytic domain, and activated PARP1 utilizes NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. Here we show that intact DNA makes contacts with the BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy (cryoEM) and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the ‘monkey-bar mechanism’ that mediates transfer of PARP1 from one DNA segment to another.

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring of the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing this process quantitatively. Here we introduce a free diffusion model that explicitly accounts for the unique topology of individual nuclei and quantitatively describes the accumulation of two test proteins, poly-ADP-ribose polymerases 1 and 2. Application of our model to other proteins will yield novel insights into the timing and mechanism of DNA repair.