Kinetic Mechanism of DNA Translocation by the RSC Molecular Motor

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning

Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC

RSC, Remodel the Structure of Chromatin, is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a ‘trimeric minimal RSC’ (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein labeled double stranded DNA with a Kd of approximately 100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration dependent manner with the addition of AMP-PNP having the more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affect the affinity of DNA binding by RSCt suggests that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the low apparent processivity of DNA translocation by the enzyme.This work was supported by National Institutes of Health Grant P20 RR017708 to C.J.F. and NIH GM60415 to B.R.C. Evan Rich was supported, in part, by an Undergraduate Research Award provided by the Kansas University Honors Program

The annual Hajj pilgrimage-minimizing the risk of ill health in pilgrims from Europe and opportunity for driving the best prevention and health promotion guidelines.

Mass gatherings at religious events can pose major public health challenges, particularly the transmission of infectious diseases. Every year the Kingdom of Saudi Arabia (KSA) hosts the Hajj pilgrimage, the largest gathering held on an annual basis where over 2 million people come to KSA from over 180 countries. Living together in crowded conditions exposes the pilgrims and the local population to a range infectious diseases. Respiratory and gastrointestinal tract bacterial and viral infections can spread rapidly and affect attendees of mass gatherings. Lethal infectious disease outbreaks were common during Hajj in the 19th and 20th centuries although they have now been controlled to a great extent by the huge investments made by the KSA into public health prevention and surveillance programs. The KSA provides regular updated Hajj travel advice and health regulations through international public health agencies such as the WHO, Public Health England, the Centers for Disease Control and Prevention, and Hajj travel agencies. During the Hajj, an additional 25 000 health workers are deployed; there are eight hospitals in Makkah and Mina complete with state-of-the-art surgical wards and intensive care units made specifically available for pilgrims. All medical facilities offer high quality of care, and services are offered free to Hajj pilgrims to ensure the risks of ill health to all pilgrims and KSA residents are minimal. A summary of the key health issues that arise in pilgrims from Europe during Hajj and of the KSA Hajj guidelines, together with other factors that may play a role in reducing the risks to pilgrims and to wider global health security, is provided herein

The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C virus Helicase NS3h is Dependent on Both the Sugar and Base Moieties

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates

Novel intervention to promote COVID-19 protective behaviours among Black and South Asian communities in the UK: protocol for a mixed-methods pilot evaluation

INTRODUCTION: Culturally appropriate interventions to promote COVID-19 health protective measures among Black and South Asian communities in the UK are needed. We aim to carry out a preliminary evaluation of an intervention to reduce risk of COVID-19 comprising a short film and electronic leaflet. METHODS AND ANALYSIS: This mixed methods study comprises (1) a focus group to understand how people from the relevant communities interpret and understand the intervention's messages, (2) a before-and-after questionnaire study examining the extent to which the intervention changes intentions and confidence to carry out COVID-19 protective behaviours and (3) a further qualitative study exploring the views of Black and South Asian people of the intervention and the experiences of health professionals offering the intervention. Participants will be recruited through general practices. Data collection will be carried out in the community. ETHICS AND DISSEMINATION: The study received Health Research Authority approval in June 2021 (Research Ethics Committee Reference 21/LO/0452). All participants provided informed consent. As well as publishing the findings in peer-reviewed journals, we will disseminate the findings through the UK Health Security Agency, NHS England and the Office for Health Improvement and Disparities and ensure culturally appropriate messaging for participants and other members of the target groups

Testing for sexually transmitted infections in general practice: cross-sectional study

Background: Primary care is an important provider of sexual health care in England. We sought to explore the extent of testing for chlamydia and HIV in general practice and its relation to associated measures of sexual health in two contrasting geographical settings.Methods: We analysed chlamydia and HIV testing data from 64 general practices and one genitourinary medicine (GUM) clinic in Brent (from mid-2003 to mid-2006) and 143 general practices and two GUM clinics in Avon (2004). We examined associations between practice testing status, practice characteristics and hypothesised markers of population need (area level teenage conception rates and Index of Multiple Deprivation, IMD scores).Results: No HIV or chlamydia testing was done in 19% (12/64) of general practices in Brent, compared to 2.1% (3/143) in Avon. In Brent, the mean age of general practitioners (GPs) in Brent practices that tested for chlamydia or HIV was lower than in those that had not conducted testing. Practices where no HIV testing was done had slightly higher local teenage conception rates (median 23.5 vs. 17.4/1000 women aged 15-44, p = 0.07) and served more deprived areas (median IMD score 27.1 vs. 21.8, p = 0.05). Mean yearly chlamydia and HIV testing rates, in practices that did test were 33.2 and 0.6 (per 1000 patients aged 15-44 years) in Brent, and 34.1 and 10.3 in Avon, respectively. In Brent practices only 20% of chlamydia tests were conducted in patients aged under 25 years, compared with 39% in Avon.Conclusions: There are substantial geographical differences in the intensity of chlamydia and HIV testing in general practice. Interventions to facilitate sexually transmitted infection and HIV testing in general practice are needed to improve access to effective sexual health care. The use of routinely-collected laboratory, practice-level and demographic data for monitoring sexual health service provision and informing service planning should be more widely evaluated

Improving uptake of hepatitis B and hepatitis C testing in South Asian migrants in community and faith settings using educational interventions - a prospective descriptive study

Background Chronic viral hepatitis (CVH) is a leading contributor to the UK liver disease epidemic, with global migration from high-prevalence areas (e.g. South Asia-SA). Despite international guidance for testing high-risk groups in line with elimination targets, there is no consensus on how to achieve this. Objectives (i) Feasibility of recruiting SA migrants to view an educational film on CVH (ii) Effectiveness of the film in promoting testing, knowledge of CVH (iii) Methodological issues relevant to scale-up to randomized trial. Methods We recruited SA migrants to view the film (intervention) in community venues (primary care, religious, community), offering dried-blood spot CVH testing immediately afterwards. Pre/post-film questionnaires assessed the interventions effectiveness. Results Two hundred and nineteen first generation migrants >18yrs (53% female) were recruited to view the film;184 (84%) underwent CVH testing (HBc Ab or HCV Ab positive, demonstrating exposure in 8.5%) at the following sites: n = 112 (51%) religious, n = 98(45%) community, and primary care, n = 9 (4%). Pre (n = 173, 79%) and post (n = 154, 70%) intervention questionnaires were completed. Conclusions We demonstrate the feasibility of recruiting first generation migrants to participate in a community-based educational film, promoting CVH testing in this higher-risk group, confirming value of developing interventions to facilitate global WHO plan for targeted case finding, elimination and future randomized controlled trial. We highlight the importance of culturally relevant interventions including faith, and culturally sensitive settings appearing to minimize logistical issues effective at engaging minority groups and allowing ease of access to individuals ‘at risk’