RecA and DNA recombination: a review of molecular mechanisms

International audienceRecombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson-Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg 2+ , two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8 to 20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer-monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics

Study of recombinase variants for the development of an isothermal amplification method

Le diagnostic des maladies infectieuses et parasitaires est un problème de santé publique mondial. Un diagnostic précis aux premiers stades de l'infection est crucial pour le contrôle des épidémies, pour la prévention et la surveillance des septis et pour la lutte contre la résistance aux antibiotiques. L'Organisation Mondiale de la Santé (OMS) définit le critère "ASSURED" pour l'évaluation des dispositifs de diagnostic et leur utilisation dans des contextes où les ressources sont limitées. Dans les pays en développement, ces dispositifs, appelés dispositifs au point d'intervention (POC), doivent être abordables, sensibles, spécifiques, faciles à utiliser, rapides et robustes, ne pas nécessiter d'équipement et pouvoir être livrés à ceux qui en ont besoin. Dans ce contexte, l'objectif de cette étude est de développer une méthode d'amplification isotherme, étape centrale du diagnostic génétique, propre à bioMérieux, utilisant deux enzymes : une recombinase RecA et une ADN polymérase (RPA). RecA joue un rôle central dans la réparation de l'ADN et est un acteur principal impliqué dans la recombinaison et l'activation de la réponse SOS. Dans la première partie de l'étude, nous avons étudié l'activité de chacune des deux enzymes et nous avons étudié la compatibilité des différents composants du mélange d'amplification. Les résultats obtenus ont mis en évidence la protéine RecA comme l'élément limitant de la réaction d'amplification RPA avec notamment une inhibition de l'activité de RecA par les dNTPs. Ainsi, au cours de la deuxième partie de l'étude, nous avons étudié les propriétés biologiques et biochimiques de quatre RecA provenant de différents organismes (Escherichia coli, Dickeya dadantii, Pseudomonas aeruginosa et Deinococcus radiodurans) et de neuf variantes de RecA de E. coli afin de mettre en évidence des variantes ayant des propriétés compatibles avec l'amplification RPA et d'apporter des informations sur le lien entre la structure et la fonction des RecA. En particulier, nous avons étudié leur activité recombinogène, leur capacité à induire la réponse SOS, leur impact sur les différents mécanismes cellulaires et nous avons également recherché les propriétés biochimiques qui peuvent être utiles pour le développement d'applications biotechnologiques. Nous avons montré que la RecA de D. dadantii (DdRecA) avait une activité d'échange de brin optimale à 30°C et en présence d'un mélange de dNTP, faisant de DdRecA un bon candidat pour le développement de RPA. De plus, l'étude de la RecA de P. aeruginosa, de la RecA de D. radiodurans et de trois variantes contenant une combinaison de mutations qui, prises indépendamment, sont décrites comme améliorant la recombinaison, nous a conduit à soulever de nouvelles hypothèses sur la relation structure-fonction et sur les interactions monomère-monomère qui perturbent l'activité de la protéine dans son ensemble.The diagnosis of infectious and parasitic diseases is a global public health issue. Accurate diagnosis in the early stages of infection is crucial for the control of epidemics, for the prevention and monitoring of sepsis and for the fight against antibiotic resistance. The World Health Organisation (WHO) defines the "ASSURED" criterion for evaluating diagnostic devices and their use in resource-limited settings. In developing countries, these devices, called point-of-care (POC) devices, must be Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, with no need for Equipment and Deliverable to those who need it. In this context, the aim of this study is to develop an isothermal amplification method, central step of the genetic diagnosis, specific to bioMérieux, using two enzymes: a recombinase RecA and a DNA polymerase (RPA). RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. During the first part of the study, we studied the activity of each of the two enzymes and we studied the compatibility of the different components of the amplification mixture. The results obtained pointed the RecA protein as the limiting element of the RPA amplification reaction with, in particular, an inhibition of RecA activity by dNTPs. Thus, during the second part of the study, we studied the biological and biochemical properties of four RecA from different organisms (Escherichia coli, Dickeya dadantii, Pseudomonas aeruginosa and Deinococcus radiodurans) and nine E. coli RecA variants in order to find variants with properties compatible with RPA amplification and to bring information about the link between RecA structure and function. In particular, we studied their recombinogenic activity, their ability to induce the SOS response, their impact on the different cellular mechanisms and we also investigated the biochemical properties that may be useful for the development of biotechnological applications. We showed that D. dadantii RecA (DdRecA) had an optimum strand exchange activity at 30°C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA (EcRecA), making Dd RecA a good candidate for RPA development. Also, the study of P. aeruginosa RecA, D. radiodurans RecA and three variants that contained a combination of mutations that, taken independently, are described as improving recombination, led us to raise new hypotheses on the structure-function relationship and on the monomer-monomer interactions that perturb the activity of the protein as a whole

La evaluación colegiada de las competencias básicas en la Comunidad Autónoma de Canarias : hacia un modelo de escuela inclusiva y sostenible

Precede al título: Educación Primaria y Educación Secundaria ObligatoriaLa Ley Orgánica de Educación (LOE) introduce el concepto de «competencias básicas» como eje articulador del currículo, conectando de pleno con las reflexiones y las estrategias que se están desarrollando en otros sistemas educativos internacionales a la luz del informe Delors (1996), el documento DeSeCo (Definición y Selección de Competencias fundamentales) elaborado por la OCDE, de las evaluaciones PISA (Programa para la Evaluación Internacional del Alumnado), etc. Esta propuesta centra el foco en la dimensión formativa de la «evaluación», aspecto inacabado con la LOGSE (Ley Orgánica General del Sistema Educativo), a pesar de los esfuerzos realizados en esa dirección. Trabajar en las aulas para la consecución de las «competencias básicas» lleva ineludiblemente al problema de cómo evaluarlas de forma colegiada —cuando la propia ordenación del sistema educativo fragmenta cada una de las enseñanzas en diferentes áreas o materias— y de cómo emplear la información que proporciona esta labor para hacer valer el sentido formativo y regulador que debe tener la evaluación de las competencias básicas.Consejería de Educación y Universidades. Dirección General de Ordenación, Innovación y Promoción Educativa; Avda. Buenos Aires, 5; 38071 Tenerife; Tel. +34922592592; Fax +34922592570; [email protected]

The value of open-source clinical science in pandemic response: lessons from ISARIC

International audienc

Clinical and genetic characteristics of late-onset Huntington's disease

Background: The frequency of late-onset Huntington's disease (>59 years) is assumed to be low and the clinical course milder. However, previous literature on late-onset disease is scarce and inconclusive. Objective: Our aim is to study clinical characteristics of late-onset compared to common-onset HD patients in a large cohort of HD patients from the Registry database. Methods: Participants with late- and common-onset (30–50 years)were compared for first clinical symptoms, disease progression, CAG repeat size and family history. Participants with a missing CAG repeat size, a repeat size of ≤35 or a UHDRS motor score of ≤5 were excluded. Results: Of 6007 eligible participants, 687 had late-onset (11.4%) and 3216 (53.5%) common-onset HD. Late-onset (n = 577) had significantly more gait and balance problems as first symptom compared to common-onset (n = 2408) (P <.001). Overall motor and cognitive performance (P <.001) were worse, however only disease motor progression was slower (coefficient, −0.58; SE 0.16; P <.001) compared to the common-onset group. Repeat size was significantly lower in the late-onset (n = 40.8; SD 1.6) compared to common-onset (n = 44.4; SD 2.8) (P <.001). Fewer late-onset patients (n = 451) had a positive family history compared to common-onset (n = 2940) (P <.001). Conclusions: Late-onset patients present more frequently with gait and balance problems as first symptom, and disease progression is not milder compared to common-onset HD patients apart from motor progression. The family history is likely to be negative, which might make diagnosing HD more difficult in this population. However, the balance and gait problems might be helpful in diagnosing HD in elderly patients

Association of Country Income Level With the Characteristics and Outcomes of Critically Ill Patients Hospitalized With Acute Kidney Injury and COVID-19

Introduction: Acute kidney injury (AKI) has been identified as one of the most common and significant problems in hospitalized patients with COVID-19. However, studies examining the relationship between COVID-19 and AKI in low- and low-middle income countries (LLMIC) are lacking. Given that AKI is known to carry a higher mortality rate in these countries, it is important to understand differences in this population. Methods: This prospective, observational study examines the AKI incidence and characteristics of 32,210 patients with COVID-19 from 49 countries across all income levels who were admitted to an intensive care unit during their hospital stay. Results: Among patients with COVID-19 admitted to the intensive care unit, AKI incidence was highest in patients in LLMIC, followed by patients in upper-middle income countries (UMIC) and high-income countries (HIC) (53%, 38%, and 30%, respectively), whereas dialysis rates were lowest among patients with AKI from LLMIC and highest among those from HIC (27% vs. 45%). Patients with AKI in LLMIC had the largest proportion of community-acquired AKI (CA-AKI) and highest rate of in-hospital death (79% vs. 54% in HIC and 66% in UMIC). The association between AKI, being from LLMIC and in-hospital death persisted even after adjusting for disease severity. Conclusions: AKI is a particularly devastating complication of COVID-19 among patients from poorer nations where the gaps in accessibility and quality of healthcare delivery have a major impact on patient outcomes

Characteristics and outcomes of COVID-19 patients admitted to hospital with and without respiratory symptoms

Background: COVID-19 is primarily known as a respiratory illness; however, many patients present to hospital without respiratory symptoms. The association between non-respiratory presentations of COVID-19 and outcomes remains unclear. We investigated risk factors and clinical outcomes in patients with no respiratory symptoms (NRS) and respiratory symptoms (RS) at hospital admission. Methods: This study describes clinical features, physiological parameters, and outcomes of hospitalised COVID-19 patients, stratified by the presence or absence of respiratory symptoms at hospital admission. RS patients had one or more of: cough, shortness of breath, sore throat, runny nose or wheezing; while NRS patients did not. Results: Of 178,640 patients in the study, 86.4 % presented with RS, while 13.6 % had NRS. NRS patients were older (median age: NRS: 74 vs RS: 65) and less likely to be admitted to the ICU (NRS: 36.7 % vs RS: 37.5 %). NRS patients had a higher crude in-hospital case-fatality ratio (NRS 41.1 % vs. RS 32.0 %), but a lower risk of death after adjusting for confounders (HR 0.88 [0.83-0.93]). Conclusion: Approximately one in seven COVID-19 patients presented at hospital admission without respiratory symptoms. These patients were older, had lower ICU admission rates, and had a lower risk of in-hospital mortality after adjusting for confounders