Structural studies of the PARP-1 BRCT domain

Here 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.The 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.The DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins localized to foci of DNA damage [1]. Although PARP-1 itself lacks any DNA-repair activity, interaction with damaged DNA stimulates the NAD+-dependent poly(ADP-ribosyl)ation activity of PARP-1 [2]. The trans-poly(ADP-ribosyl)ation of target proteins, including DNA packaging proteins [3], is postulated to reduce the DNA-binding affinity of these target proteins resulting in decondensation and accumulation of DNA-repair proteins to sites on the damaged DNA [4]. Poly(ADP-ribosyl)ation also regulates various proteins involved in cell cycle control [5], apoptosis [6] and transcriptional regulation [7]. PARP-1 is itself also a target of automodification by poly(ADP-ribose) which is important for the repair of alkylatin