A Third Zinc-binding Domain of Human Poly(ADP-ribose) Polymerase-1 Coordinates DNA-dependent Enzyme Activation

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113-kDa polypeptide. Two zinc finger domains at the N terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function; however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates interdomain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form interdomain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-totail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1.

Trapping Poly(ADP-Ribose) Polymerase

“…The distinct binding modes in the CAT domain observed among the PARP1 inhibitors (Fig. 2B) combined with newly available structural data on noncatalytic domains (Langelier et al, 2008(Langelier et al, , 2011(Langelier et al, , 2012Loeffler et al, 2011;Ali et al, 2012;Hassler and Ladurner, 2012) may suggest key structural insights into the DNA-trapping activity of PARP inhibitors. The crystal structure of the "near" full-length PARP1 enzyme containing 1) zincfinger domains Zn1 and Zn3, 2) WGR domain named after a conserved Trp-Gly-Arg sequence, and 3) CAT domain consisting of HD-ARTD subdomains bound to a DNA DSB (PDB ID: 4DQY) demonstrates that DNA binding by Zn1, Zn3, and WGR domains destabilizes the CAT domain, thereby activating the PARP enzymatic activity (Langelier et al, 2012).…”

Iniparib Nonselectively Modifies Cysteine-Containing Proteins in Tumor Cells and Is Not a Bona Fide PARP Inhibitor

“…One of the properties of PARP-1 is its ability to poly-(ADP-ribosyl)ate itself (23,24). As another measure of the ability of compounds to inhibit PARP activity, we examined auto-ribosylation of purified PARP-1 in the presence of double-stranded DNA.…”

“…The PARP-1 auto-modification assay was adapted from a method reported previously using the same double-stranded DNA sequence obtained from Integrated DNA Technologies (23). Briefly, PARP-1 was incubated with DMSO, 110 mmol/L iniparib, 110 mmol/L iniparib-met, or 110 mmol/L cmpd-B for 10 minutes followed by addition of doublestranded DNA for 20 minutes.…”

Nuclear-cytoplasmic PARP-1 expression as an unfavorable prognostic marker in lymph node-negative early breast cancer: 15-year follow-up

“…PARP-1 protein comprises three main domains: i) DNA binding domain (DBD), located at the N-terminal end of the protein; ii) automodification domain (AMD) and iii) catalytic domain (CD), which spans the C-terminus of PARP-1 molecule (4-7). DBD is composed of three zinc finger motifs (FI-III/Zn1-3) that direct PARP-1 binding to DNA (FI/Zn1 and FII/Zn2) and are involved in interdomain and protein-protein interactions, which is crucial for DNA-dependent enzyme activation (FIII/Zn3) (8,9). AMD is important for the proper biological functioning, and encompasses approximately 15 fragments (mainly glutamic acid) which are sites of PAR chains attachment.…”