Nucleic-acid detection via isothermal amplification and collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative polymerase chain reaction (qPCR). Here, we report the clinical validation of the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) -the virus that causes COVID-19 (coronavirus disease 2019) -in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.

Nucleotide excision repair (NER) is used by all organisms to eliminate DNA lesions. We determined the structure of the Geobacillus stearothermophilus UvrA-UvrB complex, the damage-sensor in bacterial NER and a new structure of UvrA. We observe that the DNA binding surface of UvrA, previously found in an open shape that binds damaged DNA, also exists in a closed groove shape compatible with native DNA only. The sensor contains two UvrB molecules that flank the UvrA dimer along the predicted path for DNA, ~80 Å from the lesion. We show that the conserved signature domain II of UvrA mediates a nexus of contacts among UvrA, UvrB and DNA. Further, in our new structure of UvrA, this domain adopts an altered conformation while an adjacent nucleotide binding site is vacant. Our findings raise unanticipated questions about NER and also suggest a revised picture of its early stages.

Background The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. Results The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z’ factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. Conclusion The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.

Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA⅐UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for fulllength UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a diverse set of lesions. In bacteria, the initial steps are carried out by three proteins: UvrA, UvrB, and UvrC. The UvrA⅐UvrB complex conducts surveillance of DNA and recognizes damage. Having located a lesion, UvrA "loads" UvrB onto the DNA at the damaged sites and then dissociates. Damage searching, formation of the UvrB⅐DNA "preincision" complex, and dissociation of UvrA are regulated by ATP (1). UvrB subsequently recruits the endonuclease UvrC, which catalyzes incisions on either side of the lesion (2, 3). Following incision, UvrC and the damage-containing oligonucleotide are removed by UvrD (helicase II), whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for repair synthesis. Sealing of the single-stranded nick completes the repair process and restores the original DNA sequence (4).Since its discovery more than 40 years ago, bacterial nucleotide excision repair has been extensively studied, resulting in a large body of work that describes the protein components and the details of how they operate. Notwithstanding the trove of genetic and biochemical data, several key questions remain unanswered. For example, how does the same set of proteins handle a diverse set of lesions while maintaining specificity? How do UvrA and UvrB cooperate during damage recognition, and what is the precise role of ATP? Ongoing studies in the field, including those described below, aim to address these issues.Recently, we reported the structure of Geobacillus stearothermophilus UvrA and the identification of binding sites for DNA and UvrB (5). We also established that the identified UvrB-binding domain is necessary and sufficient to mediate the UvrA-UvrB interaction and that the isolated interaction domains of UvrA (5) and UvrB (6) bind to each other in solution.To understand the interaction between UvrA and UvrB, we have determined the crystal structure of the complex between the two isolated interaction domains. The structure revealed that UvrA-UvrB interaction interface is largely polar, mediated by several highly conserved charged residues. Site-directed mutagenesis and biochemical characterization of the mutant proteins confirmed the importance of the observed interactions. Based on the interaction domain complex structure, we have constructed a structural model for the full-length UvrA⅐UvrB ensemble an...