Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

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.

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.

Improved design method for biosecurity surveillance and early detection of non-indigenous rats

A recent advance in biosecurity surveillance design aims to benefit island conservation through early and improved detection of incursions by non-indigenous species. The novel aspects of the design are that it achieves a specified power of detection in a cost-managed system, while acknowledging heterogeneity of risk in the study area and stratifying the area to target surveillance deployment. The design also utilises a variety of surveillance system components, such as formal scientific surveys, trapping methods, and incidental sightings by non-biologist observers. These advances in design were applied to black rats (Rattus rattus) representing the group of invasive rats including R. norvegicus, and R. exulans, which are potential threats to Barrow Island, Australia, a high value conservation nature reserve where a proposed liquefied natural gas development is a potential source of incursions. Rats are important to consider as they are prevalent invaders worldwide, difficult to detect early when present in low numbers, and able to spread and establish relatively quickly after arrival. The ‘exemplar’ design for the black rat is then applied in a manner that enables the detection of a range of non-indigenous species of rat that could potentially be introduced. Many of the design decisions were based on expert opinion as data gaps exist in empirical data. The surveillance system was able to take into account factors such as collateral effects on native species, the availability of limited resources on an offshore island, financial costs, demands on expertise and other logistical constraints. We demonstrate the flexibility and robustness of the surveillance system and discuss how it could be updated as empirical data are collected to supplement expert opinion and provide a basis for adaptive management. Overall, the surveillance system promotes an efficient use of resources while providing defined power to detect early rat incursions, translating to reduced environmental, resourcing and financial costs.

The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18–30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.

Ecological aspects of biosecurity surveillance design for the detection of multiple invasive animal species

Complex surveillance problems are common in biosecurity, such as prioritizing detection among multiple invasive species, specifying risk over a heterogeneous landscape, combining multiple sources of surveillance data, designing for specified power to detect, resource management, and collateral effects on the environment. Moreover, when designing for multiple target species, inherent biological differences among species result in different ecological models underpinning the individual surveillance systems for each. Species are likely to have different habitat requirements, different introduction mechanisms and locations, require different methods of detection, have different levels of detectability, and vary in rates of movement and spread. Often there is a further challenge of a lack of knowledge, literature, or data, for any number of the above problems. Even so, governments and industry need to proceed with surveillance programs which aim to detect incursions in order to meet environmental, social and political requirements. We present an approach taken to meet these challenges in one comprehensive and statistically powerful surveillance design for non-indigenous terrestrial vertebrates on Barrow Island, a high conservation nature reserve off the Western Australian coast. Here, the possibility of incursions is increased due to construction and expanding industry on the island. The design, which includes mammals, amphibians and reptiles, provides a complete surveillance program for most potential terrestrial vertebrate invaders. Individual surveillance systems were developed for various potential invaders, and then integrated into an overall surveillance system which meets the above challenges using a statistical model and expert elicitation. We discuss the ecological basis for the design, the flexibility of the surveillance scheme, how it meets the above challenges, design limitations, and how it can be updated as data are collected as a basis for adaptive management.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T cells

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

An examination of tax-deductible donations made by individual Australian taxpayers in 2008-09, ACPNS Working Paper 54

Each year, The Australian Centre for Philanthropy and Nonprofit Studies (CPNS) at Queensland University of Technology (QUT) collects and analyses statistics on the amount and extent of tax-deductible donations made and claimed by Australians in their individual income tax returns to deductible gift recipients (DGRs). The information presented below is based on the amount and type of tax-deductible donations made and claimed by Australian individual taxpayers to DGRs for the period 1 July 2008 to 30 June 2009. This information has been extracted mainly from the Australian Taxation Office's (ATO) publication Taxation Statistics 2008-09. The 2008-09 report is the latest report that has been made publicly available. It represents information in tax returns for the 2008-09 year processed by the ATO as at 31 October 2010.

From allure to ethics : design as a ‘creative industry'

In this chapter I look at the question of design and ethics in a situation where design as a set of related economic practices has been re-positioned as a key identifier for a new kind of ideas-driven industry - the creative industries. Previously marginal to cultural policy in the form of applied arts, in the creative industries agenda design became a privileged meeting place, or indeed broker, for art and industry, economy and commerce. I explore the new kinds of ethical challenges this brings.