Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here, we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a ‘monkey bar’ mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA.

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 6Â 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.

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Mammalian CXXC finger protein 1 (Cfp1) is a DNA-binding protein that is a component of the Setd1 histone methyltransferase complexes and is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a loss of histone H3-Lys4 tri-methylation (H3K4me3) at many CpG islands, and a mis-localization of this epigenetic mark to heterochromatic sub-nuclear domains.Furthermore, these cells fail to undergo cellular differentiation in vitro. These defects are rescued upon introduction of a Cfp1-expression vector. Cfp1 contains an N-terminal plant homeodomain (PHD), a motif frequently observed in chromatin associated proteins that functions as a reader module of histone marks. Here, we report that the Cfp1 PHD domain directly and specifically binds to histone H3K4me1/me2/me3 marks. Introduction of individual mutations at key Cfp1 PHD residues (Y28, D44, or W49) ablates this histone interaction both in vitro and in vivo. The W49A point mutation does not affect the ability of Cfp1 to rescue appropriate restriction of histone H3K4me3 to euchromatic subnuclear domains or in vitro cellular differentiation in Cfp1-null ES cells. Similarly, a mutated form of Cfp1 that lacks DNA-binding activity (C169A) rescues in vitro cellular differentiation. However, rescue of Cfp1-null ES cells with a double mutant form of Cfp1 (W49A, C169A) results in partially defective in vitro differentiation. These data define the Cfp1 PHD domain as a reader of histone H3K4me marks and provide evidence that this activity is involved in the regulation of lineage commitment in ES cells.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.