Structural studies of the PARP-1 BRCT domain

Abstract

BackgroundPoly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins localized to foci of DNA damage. Upon activation by encountering nicked DNA, the PARP-1 mediated trans-poly(ADP-ribosyl)ation of DNA binding proteins occurs, facilitating access and accumulation of DNA repair factors. PARP-1 also auto-(ADP-ribosyl)ates its central BRCT-containing domain forming part of an interaction site for the DNA repair scaffolding protein X-ray cross complementing group 1 protein (XRCC1). The co-localization of XRCC1, as well as bound DNA repair factors, to sites of DNA damage is important for cell survival and genomic integrity.ResultsHere we present the solution structure and biophysical characterization of the BRCT domain of rat PARP-1. The PARP-1 BRCT domain has the globular α/β fold characteristic of BRCT domains and has a thermal melting transition of 43.0°C. In contrast to a previous characterization of this domain, we demonstrate that it is monomeric in solution using both gel-filtration chromatography and small-angle X-ray scattering. Additionally, we report that the first BRCT domain of XRCC1 does not interact significantly with the PARP-1 BRCT domain in the absence of ADP-ribosylation. Moreover, none of the interactions with other longer PARP-1 constructs which previously had been demonstrated in a pull-down assay of mammalian cell extracts were detected.ConclusionsThe PARP-1 BRCT domain has the conserved BRCT fold that is known to be an important protein:protein interaction module in DNA repair and cell signalling pathways. Data indicating no significant protein:protein interactions between PARP-1 and XRCC1 likely results from the absence of poly(ADP-ribose) in one or both binding partners, and further implicates a poly(ADP-ribose)-dependent mechanism for localization of XRCC1 to sites of DNA damage.

Primary Identification, Biochemical Characterization, and Immunologic Properties of the Allergenic Pollen Cyclophilin Cat r 1

“…Resonance assignments have been deposited in the BioMagResBank database, entry 19432. The solution structure was determined as described elsewhere (33). The 15 N-1 H HSQC spectrum of the backbone amides was unusually disperse, making the backbone assignments facile (see below).…”

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).…”

Coordination of DNA single strand break repair

“…In addition to PARylation of acceptor proteins, PARP1 is also able to directly interact with protein partners via a BRCT domain located at residues 389-487 within the central AD [96]. The PARP1 BRCT domain shares sequence homology with the highly conserved BRCA1 carboxyl-terminal (BRCT) domain superfamily that mediates protein interactions with a large number of DNA repair factors [97].…”