Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding

Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3′–5′ polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (∼40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from ∼40 to ∼150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from ∼45 to ∼10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer

Competência ortográfica e metafonológica: influências e correlações na leitura e escrita de escolares da 4ª série Orthographic and metaphonological competences: influences and correlations with reading and writing abilities of 4th grade students

OBJETIVO: Estudar o desempenho de escolares da 4ª série em tarefas de leitura e escrita e de consciência fonológica. MÉTODOS: Trinta e dois escolares de 4ª série (50,0% meninas), entre nove e 11 anos, foram avaliados segundo os procedimentos: anamnese, avaliação da leitura, escrita (54 itens linguísticos) e consciência fonológica. Foram divididos em: GPI: escreveram e, após, leram em voz alta; GPII: leram em voz alta e, após, escreveram; GCI: apenas escreveram; GCII: apenas leram. RESULTADOS: Na escrita, GPII apresentou as maiores médias de acerto, sendo que GPI, GPII e GCI apresentaram melhor desempenho para as palavras frequentes, seguido das pseudopalavras e das de baixa frequência. Na leitura, os desempenhos dos GPI, GPII e GCII foram semelhantes, sendo que GPI e GPII apresentaram mais acertos para as pseudopalavras, seguido das de alta frequência. A correlação entre consciência fonológica e escrita mostrou-se moderada e entre consciência fonológica e leitura e entre escrita e leitura foi bem fraca. CONCLUSÕES: A consciência fonológica mostrou moderada correlação com a escrita e baixa com a leitura; a característica psicolinguística baixa familiaridade determinou as maiores médias de erro, tanto na leitura quanto na escrita.<br>PURPOSE: To study the performance of 4th grade students on reading, writing and phonological awareness tasks. METHODS: Thirty-two 4th grade children (50% of each gender), with ages between nine and 11 years, were evaluated according to the following procedure: anamnesis, reading and writing task (54 linguistic tokens), and phonological awareness task. The students were divided into two research groups and two comparison groups: GPI: carried out the writing task first, and then the reading task; GPII: completed the reading, followed by the writing task; GCI: completed only the writing items; GCII: completed only the reading items. RESULTS: In the writing task, GPII showed the highest rates of correct answers, and GPI, GPII and GCI had better performances in writing high frequency words, followed by pseudowords and low frequency words. The reading evaluation showed that the performances of GPI, GPII and GCII were similar, and GPI and GPII had more correct answers for pseudowords, followed by high frequency words. The correlation between phonological awareness and writing was moderate, while correlations between phonological awareness and reading, and writing and reading were very weak. CONCLUSION: Phonological awareness was moderately correlated to the performance on the writing task, and weakly correlated to the performance on the reading task. The psycholinguistic feature low familiarity determined the highest error averages, both in reading and writing tasks

ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit