Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase

Relationship between e-cigarette point of sale recall and e-cigarette use in secondary school children: a cross-sectional study

Background There has been a rapid increase in the retail availability of e-cigarettes in the UK and elsewhere. It is known that exposure to cigarette point-of-sale (POS) displays influences smoking behaviour and intentions in young people. However, there is as yet no evidence regarding the relationship between e-cigarette POS display exposure and e-cigarette use in young people. Methods This cross sectional study survey was conducted in four high schools in Scotland. A response rate of 87% and a total sample of 3808 was achieved. Analysis was by logistic regression on e-cigarette outcomes with standard errors adjusted for clustering within schools. The logistic regression models were adjusted for recall of other e-cigarette adverts, smoking status, and demographic variables. Multiple chained imputation was employed to assess the consistency of the findings across different methods of handling missing data. Results Adolescents who recalled seeing e-cigarettes in small shops were more likely to have tried an e-cigarette (OR 1.92 99% CI 1.61 to 2.29). Adolescents who recalled seeing e-cigarettes for sale in small shops (OR 1.80 99% CI 1.08 to 2.99) or supermarkets (OR 1.70 99% CI 1.22 to 2.36) were more likely to intend to try them in the next 6 months. Conclusions This study has found a cross-sectional association between self-reported recall of e-cigarette POS displays and use of, and intention to use, e-cigarettes. The magnitude of this association is comparable to that between tobacco point of sale recall and intention to use traditional cigarettes in the same sample. Further longitudinal data is required to confirm a causal relationship between e-cigarette point of sale exposure and future use in young people.Publisher PDFPeer reviewe

Splitting, joining and cutting : mechanistic studies of enzymes that manipulate DNA

Electronic version does not contain associated previously published materialDNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea

Splitting, joining and cutting : mechanistic studies of enzymes that manipulate DNA

DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

MULTI-CENTER TRIAL WITH PROTOCOLS AND PROTOTYPE TEST OBJECTS FOR THE ASSESSMENT OF MRI EQUIPMENT

International audienc

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed

Structure of the DNA Repair Helicase XPD

SummaryThe XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 Å crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans

Giving the silent majority a stronger voice? Initiatives to empower Muslim women as part of the UK's 'War on Terror'

This article provides a gendered analysis of the 'War on Terror' in the UK context. Specifically it looks at initiatives to empower Muslim women, which were part of New Labour's Preventing Violent Extremism (PVE) agenda, the impetus for which stemmed from the idea that, as 'the silent majority', women need to be given a 'stronger voice'. Based on analysis of qualitative interviews, this article situates these initiatives within a broader policy landscape of debates on multiculturalism, community cohesion and Britishness. It explores interviewees' understandings of Muslim women's silence in relation to those suggested by policy discourse, considering the ways in which the state's attempt to 'give voice' worked in practice. I argue that the operation of such initiatives continued to constrain Muslim women's voices, restricting 'voice' to a narrow range of speakers speaking about a narrow range of issues