The RIR motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair

The scaffold protein X-ray repair cross-complementing 1 (XRCC1)interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair(SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase(PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay(MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylationdependent interaction site in XRCC1 and a fork-head associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved REV1-interacting (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified highaffinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair1,2. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP3,4,5 and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide

CsA can induce DNA double-strand breaks: implications for BMT regimens particularly for individuals with defective DNA repair

Several human disorders mutated in core components of the major DNA double-strand break (DSB) repair pathway, non-homologous end joining (NHEJ), have been described. Cell lines from these patients are characterized by sensitivity to DSB-inducing agents. DNA ligase IV syndrome (LIG4) patients specifically, for unknown reasons, respond particularly badly following treatment for malignancy or BMT. We report the first systematic evaluation of the response of LIG4 syndrome to compounds routinely employed for BMT conditioning. We found human pre-B lymphocytes, a key target population for BMT conditioning, when deficient for DNA ligase IV, unexpectedly exhibit significant sensitivity to CsA the principal prophylaxis for GVHD. Furthermore, we found that CsA treatment alone or in combination with BU and fludarabine resulted in increased levels of DSBs specifically in LIG4 syndrome cells compared to wild-type or Artemis-deficient cells. Our study shows that CsA can induce DSBs and that LIG4 syndrome patient's fail to adequately repair this damage. These DSBs likely arise as a consequence of DNA replication in the presence of CsA. This work has implications for BMT and GVHD management in general and specifically for LIG4 syndrome

Primary physical education, coaches and continuing professional development

This is an Author's Accepted Manuscript of an article published in Sport, Education and Society, 16(4), 485 - 505, 2011, copyright @ Taylor & Francis, available online at: http://www.tandfonline.com/10.1080/13573322.2011.589645.Physical education (PE) in primary schools has traditionally been taught by qualified primary teachers. More recently, some teaching of PE in primary schools has been undertaken by coaches (mostly football coaches). These coaches hold national governing body awards but do not hold teaching qualifications. Thus, coaches may not be adequately prepared to teach PE in curriculum time. The purpose of this study was to evaluate the perceptions of a group of community-based football coaches working in primary schools for the impact of a Continuing Professional Development (CPD) programme on their ability to undertake ‘specified work’ to cover PE in primary schools. The programme focused on four areas identified as important to enable coaches to cover specified work: short- and medium-term planning, pedagogy, knowledge of the curriculum and reflection. Results showed that for the majority of coaches the CPD programme had made them more aware of the importance of these four areas and had helped to develop their knowledge and ability to put this into practice in covering planning, preparation and assessment time. However, further input is still required to develop coaches’ knowledge and understanding in all four areas, but especially their curriculum knowledge, as well as their ability to put these into practice consistently. These findings are discussed in relation to the implications of employing coaches to cover the teaching of PE in primary schools and, if employed, what CPD coaches need to develop the necessary knowledge, skill and understanding for covering specified work in schools

The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs

The genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1

Defective responses to DNA single strand breaks underlie various neurodegenerative diseases. However, the exact role of this repair pathway during the development and maintenance of the nervous system is unclear. Using murine neural-specific inactivation of Xrcc1, a factor that is critical for the repair of DNA single strand breaks, we found a profound neuropathology that is characterized by the loss of cerebellar interneurons. This cell loss was linked to p53-dependent cell cycle arrest and occurred as interneuron progenitors commenced differentiation. Loss of Xrcc1 also led to the persistence of DNA strand breaks throughout the nervous system and abnormal hippocampal function. Collectively, these data detail the in vivo link between DNA single strand break repair and neurogenesis and highlight the diverse consequences of specific types of genotoxic stress in the nervous system

Towards a synthesized critique of neoliberal biodiversity conservation

During the last three decades, the arena of biodiversity conservation has largely aligned itself with the globally dominant political ideology of neoliberalism and associated governmentalities. Schemes such as payments for ecological services are promoted to reach the multiple ‘wins’ so desired: improved biodiversity conservation, economic development, (international) cooperation and poverty alleviation, amongst others. While critical scholarship with respect to understanding the linkages between neoliberalism, capitalism and the environment has a long tradition, a synthesized critique of neoliberal conservation - the ideology (and related practices) that the salvation of nature requires capitalist expansion - remains lacking. This paper aims to provide such a critique. We commence with the assertion that there has been a conflation between ‘economics’ and neoliberal ideology in conservation thinking and implementation. As a result, we argue, it becomes easier to distinguish the main problems that neoliberal win-win models pose for biodiversity conservation. These are framed around three points: the stimulation of contradictions; appropriation and misrepresentation and the disciplining of dissent. Inspired by Bruno Latour’s recent ‘compositionist manifesto’, the conclusion outlines some ideas for moving beyond critique