INTS3 controls the hSSB1-mediated DNA damage response

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3–MISE–hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function.

Involvement of Exo1b in DNA damage-induced apoptosis

Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair1. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer1. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with singleand double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 A° in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short b-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Drawing on imagination : primary students' ideal learning environments

Research has established a close relationship between learning environments and learning outcomes (Department of Education and Early Childhood Development, Victoria, 2008; Woolner, Hall, Higgins, McCaughey & Wall, 2007) yet little is known about how students in Australian schools imagine the ways that their learning environments could be improved to enhance their engagement with the processes and content of education and children are rarely consulted on the issue of school design (Rudduck & Flutter, 2004). Currently, school and classroom designers give attention to operational matters of efficiency and economy, so that architecture for children’s education is largely conceived in terms of adult and professional needs (Halpin, 2007). This results in the construction of educational spaces that impose traditional teaching and learning methods, reducing the possibilities of imaginative pedagogical relationships. Education authorities may encourage new, student-centred pedagogical styles, such as collaborative learning, team-teaching and peer tutoring, but the spaces where such innovations are occurring do not always provide the features necessary to implement these styles. Heeding the views of children could result in the creation of spaces where more imaginative pedagogical relationships and student-centred pedagogical styles can be implemented. In this article, a research project conducted with children in nine Queensland primary schools to investigate their ideas of the ideal ‘school’ is discussed. Overwhelmingly, the students’ work emphasised that learning should be fun and that learning environments should be eco-friendly places where their imaginations can be engaged and where they learn from and in touch with reality. The children’s imagined schools echo ideas that have been promoted over many decades by progressive educators such as John Dewey (1897, in Provenzo, 2006) (“experiential learning”), AS Neill (in Cassebaum, 2003) (Summerhill school) and Ivan Illich (1970) (“deschooling”), with a vast majority of students suggesting that, wherever possible, learning should take place away from classrooms and in environments that support direct, hands-on learning.

Procurement strategies : a relationship-based approach

Construction has been an industry characterised by disputes, fierce competitiveness and fragmentation - all major obstacles to development. Now, however, a relationship-based approach to project procurement, through partnering and alliancing, aims to bring about a fundamental change. ----- ----- ----- This book addresses the critical relationship issues for a more collaborative and sustainable construction industry. It looks at how project procurement and project alliancing partner selection works, and how risk and crisis resolution are managed. It provides readers with guidance and models on how to put a relationship-based approach to procurement into practice, drawing on specific prototypes from an actual, successful project that can be adapted.

Construction project delivery and industry skills : crafting a better fit

The construction industry should be a priority to all governments because it impacts economically and socially on all citizens. Sector turnover in industrialised economies typically averages 8-12% of GDP. Further, construction is critical to economic growth. Recent Australian studies estimate that a 10% gain in efficiency in construction translates to a 2.5% increase in GDP ----- ----- Inefficiencies in the Australian construction industry have been identified by a number of recent studies modelling the building process. They have identified potential savings in time of between 25% and 40% by reducing non-value added steps in the process. A culture of reform is now emerging in the industry – one in which alternate forms of project delivery are being trial. ----- ----- Government and industry have identified Alliance Contracting as a means to increase efficiency in the construction industry as part of a new innovative procurement environment. Alliance contracting requires parties to form relationships and work cooperatively to provide a more complete service. This is a significant cultural change for the construction industry, with its well-known adversarial record in traditional contracting. Alliance contracts offer enormous potential benefits, but the Australian construction industry needs to develop new skills to effectively participate in the new relationship environment. ----- ----- This paper describes a collaborative project identifying skill needs for clients and construction professionals to more effectively participate in an increasingly sophisticated international procurement environment. The aim of identifying these skill needs is to assist industry, government, and skill developers to prepare the Australian construction workforce for the future. The collaborating Australian team has been fortunate to secure the Australian National Museum in Canberra as its live case study. The Acton Peninsula Development is the first major building development in the world awarded on the basis of a joint alliance contract.