Domain C of Human Poly(ADP-ribose) Polymerase-1 Is Important for Enzyme Activity and Contains a Novel Zinc-Ribbon Motif^,

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins using NAD (+) as a substrate. In this work, NMR methods were used to determine the solution structure of human PARP-1 protein. Domain C was found to contain a zinc-binding motif of three antiparallel beta-strands with four conserved cysteines positioned to coordinate the metal ligand, in addition to a helical region. The zinc-binding motif is structurally reminiscent of the "zinc-ribbon" fold, but with a novel spacing between the conserved cysteines (CX2CX12CX 9C). Domain C alone does not appear to bind to DNA. Interestingly, domain C is essential for PARP-1 activity, since a mixture containing nicked DNA and the PARP-1 ABDEF domains has only basal enzymatic activity, while the addition of domain C to the mixture initiated NAD (+) hydrolysis and the formation of poly(ADP-ribose), as detected by an NMR-based assay and autoradiography. The structural model for domain C in solution provides an important framework for further studies aimed at improving our understanding of how the various domains within the complex PARP-1 enzyme play their respective roles in regulating the enzyme activity when cells are under conditions of genotoxic stress.

The Zn3 Domain of Human Poly(ADP-ribose) Polymerase-1 (PARP-1) Functions in Both DNA-dependent Poly(ADP-ribose) Synthesis Activity and Chromatin Compaction

“…The Zn3 dimer interface mutants map to the N-terminal helical region when viewed in the context of the monomeric NMR structure (31). The crystal structure and the NMR structure of the Zn3 domain are overall very similar (Fig.…”

“…The first region of interest (residue 290 to 332) forms a zinc-binding motif reminiscent of a zinc-ribbon fold ( Fig. 2A) (30,31). Zinc-ribbon folds are often involved in mediating protein-protein interactions (32).…”

“…This was based on the fact that a fragment, including the first three zinc fingers (Zn1-Zn2-Zn3; residues 1-366), could restore DNA-dependent activity of a BRCA1 C-terminal WGR-CAT fragment of PARP-1 (residues 379 -1014), whereas a fragment lacking the Zn3 domain could not (Zn1-Zn2; residues 1-234), despite having a robust affinity for DNA. Thus, deletion of the Zn3 domain dramatically compromises PARP-1 activity without compromising DNA binding affinity (30,31). Previous mutagenesis experiments targeting the Zn3 domain are likely to have disrupted the fold of the Zn3 domain (30, 36) and therefore are comparable Mutation of residues colored red led to a defect in DNA-dependent activity, although mutation of residues colored green had no affect on DNA-dependent activity.…”

“…1, A and B) (30). The Zn3 domain is critically required for DNA-dependent PARP-1 activity (30,31). The Zn3 domain structure consists of a unique type of zinc-ribbon fold and an ␣-helical N-terminal region (Fig.…”

A Wheat SIMILAR TO RCD-ONE Gene Enhances Seedling Growth and Abiotic Stress Resistance by Modulating Redox Homeostasis and Maintaining Genomic Integrity

“…In Vitro Protein-DNA Binding Assay (Dot Blotting) Dot blotting was performed as described previously with slight modifications (Tao et al, 2008). Ta-sro1-His 6 , Ta-SRO1-His 6 , At-PARP1-His 6 , and Ta-ACO1-His 6 proteins were first blotted onto a polyvinylidene difluoride membrane.…”