Binding dynamics of a monomeric SSB protein to DNA : a single-molecule multi-process approach

People Programme of the European Union’s Seventh Framework Programme [REA 334496 to B.E.B.]; Leonardo da Vinci European Union Programme (to M.F.G.); Wellcome Trust [099149/Z/12/Z, 091825/Z/10/Z]. Funding for open access charge: Wellcome Trust; University of St Andrews.Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between highdensity binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.Publisher PDFPeer reviewe

Asymmetric base-pair opening drives helicase unwinding dynamics

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix

Mechanism of DNA loading by the DNA repair helicase XPD

Funding: Welcome Trust Programme Grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust [099149/Z/12/Z]; Royal Society Wolfson Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.Publisher PDFPeer reviewe

Emotion-Recognition Using Smart Watch Sensor Data: Mixed-Design Study

Background: Research in psychology has shown that the way a person walks reflects that person’s current mood (or emotional state). Recent studies have used mobile phones to detect emotional states from movement data. Objective: The objective of our study was to investigate the use of movement sensor data from a smart watch to infer an individual’s emotional state. We present our findings of a user study with 50 participants. Methods: The experimental design is a mixed-design study: within-subjects (emotions: happy, sad, and neutral) and between-subjects (stimulus type: audiovisual “movie clips” and audio “music clips”). Each participant experienced both emotions in a single stimulus type. All participants walked 250 m while wearing a smart watch on one wrist and a heart rate monitor strap on the chest. They also had to answer a short questionnaire (20 items; Positive Affect and Negative Affect Schedule, PANAS) before and after experiencing each emotion. The data obtained from the heart rate monitor served as supplementary information to our data. We performed time series analysis on data from the smart watch and a t test on questionnaire items to measure the change in emotional state. Heart rate data was analyzed using one-way analysis of variance. We extracted features from the time series using sliding windows and used features to train and validate classifiers that determined an individual’s emotion. Results: Overall, 50 young adults participated in our study; of them, 49 were included for the affective PANAS questionnaire and 44 for the feature extraction and building of personal models. Participants reported feeling less negative affect after watching sad videos or after listening to sad music, P<.006. For the task of emotion recognition using classifiers, our results showed that personal models outperformed personal baselines and achieved median accuracies higher than 78% for all conditions of the design study for binary classification of happiness versus sadness. Conclusions: Our findings show that we are able to detect changes in the emotional state as well as in behavioral responses with data obtained from the smartwatch. Together with high accuracies achieved across all users for classification of happy versus sad emotional states, this is further evidence for the hypothesis that movement sensor data can be used for emotion recognition

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed

<p>Abstract</p> <p>Background</p> <p>Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed.</p> <p>Methods</p> <p>46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity.</p> <p>Results</p> <p>Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, R_t = 5.7 ± 1.4, H_o = 0.63 ± 0.19 and H_e = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (F_is = 0.083, <it>P</it> < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (F_st = 0.028, <it>P</it> > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity.</p> <p>Conclusions</p> <p>Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.</p

Evaluation of organic, conventional and intensive beef farm systems: health, management and animal production

The overall aim of the present study was to analyse and compare organic beef cattle farming in Spain with intensive and conventional systems. An on-farm study comparing farm management practices and animal health was carried out. The study also focussed on a slaughterhouse analysis by comparing impacts on the safety and quality of the cattle products. Twenty-four organic and 26 conventional farms were inspected, and farmers responded to a questionnaire that covered all basic data on their husbandry practices, farm management, veterinary treatments and reproductive performance during 2007. Furthermore, data on the hygiene and quality of 244, 2596 and 3021 carcasses of calves from organic, intensive and conventional farms, respectively, were retrieved from the official yearbook (2007) of a slaughterhouse. Differences found between organic and conventional farms across the farm analysis did not substantially reflect differences between both farm types in the predominant diseases that usually occur on beef cattle farms. However, calves reared organically presented fewer condemnations at slaughter compared with intensive and to a lesser extent with conventionally reared calves. Carcass performance also reflected differences between farm type and breed and was not necessarily better in organic farmsThis study was supported by the Xunta de Galicia (Spain;PGIDT02RA6261001PR) and I.B.P. received a research fellow-ship (AP2003-3835) from the Ministry of Science of SpainS

Vector-like Singlet Quarks: a Roadmap

We review the theory and phenomenology of isosinglet vector-like quarks (VLQs). In recent years, interest in VLQs has been increasing, due to their contributions to new physics effects that can be tested in experiments at LHC and High-Luminosity LHC. The similarities of models with isosinglet VLQs and the seesaw framework in the leptonic sector are pointed out. The existence of VLQs leads to flavour-changing neutral currents at tree level and deviations from unitarity of the CKM matrix, introducing rich phenomenological implications. These new effects are naturally suppressed by the masses of the new quarks, that are constrained to be above the electroweak scale. In addition, striking new effects can be achieved with the inclusion of an extra complex scalar singlet. Such a minimal extension of the SM can give rise to new sources of CP violation with profound theoretical implications, allowing for a solution to the strong CP problem and a possible explanation for the baryon asymmetry of the Universe. We list and explain strong motivations to consider this class of models. We also briefly review how models with VLQs can be matched to the SM effective field theory (SMEFT). A detailed analysis of flavour observables that can be affected by the presence of VLQs is presented. Current bounds from collider searches of VLQs are summarized. We point out that the discovery of VLQs can be within the reach of present or future colliders being planned.Comment: 109 pages, 7 figures, 7 tables. Work intended as a review. Comments, suggestions and missing references are welcome