Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage-induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.

Fig. 1. Overview of the PARP-1/DNA crystal structure

(A) Modular domain architecture of human PARP-1. (B) Colorimetric assay of PARP-1 DNA-dependent automodification using the indicated domain combinations (see also Fig. S1 A,B). (C) Surface representation of the PARP-1/DNA structure. (D) A 90° rotation was applied to the view in C. (E) The Zn2 and BRCT domains (light grey) were manually positioned on the PARP-1/DNA structure using the structures of Zn2 and BRCT (PDB codes 3odc 2cok, respectively). The arrow accents the close proximity of AD and CAT.

Fig. 2. PARP-1 forms a multi-domain DNA binding interface

Zn1, Zn3, and WGR collectively form an interface with DNA. (A) Key Zn1–, Zn3–, and WGR–DNA contacts are highlighted. (B) Schematic representation of PARP-1 contacts with DNA.

Fig. 3. DNA-induced interdomain contacts are critical for DNA-dependent PARP-1 activation

(A) Surface representation of Zn1 and Zn3 bound to DNA. WGR and CAT have been omitted for clarity. (B) The Zn1 base-stacking loop, the Zn3 extended loop, and the 5'-terminated DNA strand form a binding site for WGR. (C) The Zn1–WGR–HD interface. (D) The HD-WGR–Zn3 interface. (E) The Zn3–Zn1 interface. Residues targeted for mutagenesis are labeled (yellow). Residues identified in a random screen for inactive mutants (20) are labeled (green). (F) SDS-PAGE assay of DNA-dependent PARP-1 automodification activity. WT and the indicated mutants were monitored for a shift in migration due to the covalent addition of PAR.

Fig. 4. Distortion of the HD structure modulates PARP-1 catalytic activity

(A) Cα trace of CAT structure in the absence of DNA and regulatory domains (PDB code 1a26; blue), and CAT in the PARP-1/DNA structure (yellow/brown). (B) Detailed view of Leu698 and Leu701 contributions to the HD hydrophobic interior of the isolated CAT (blue), and their re-positioning in the PARP-1/DNA structure (yellow with green side chains). (C) Ribbon representation of CAT in the PARP-1/DNA complex. Residues mutated are drawn in pink and labeled: HD interior hydrophobic core (white), HD exterior (black). The position of NAD⁺ was modeled for reference. (D) Radioactive assay of DNA-independent PARP-1 automodification using ³²P-NAD⁺. (E) Colorimetric assay of DNA-independent PARP-1 automodification. Values represent quantification of the 90-minute time point of a time course experiment (Fig. S7) and are an average of three independent experiments with associated standard deviations. (F) Relative thermal stability of PARP-1 mutants obtained by differential scanning fluorimetry. Changes in thermal stability (ΔT_M) were calculated as shown. (G) Relative change in thermal stability upon DNA binding. ΔT_M were calculated as shown.