Serendipity reveals the function and physiological role of a large family of proteins.

Microbial metabolism involves a complex set of interactions between metabolic pathways that include proteins of both known and uncharacterized function. While investigating the physiological strategy used by actinomycetes with two RpoB paralogs, Damiano et al uncovered the endonuclease activity of a member of the Rid family. While this finding was peripheral to the original question posed by the authors, it has considerable significance. The study by Damiano et al highlights how unexpected, but fundamental, information can be gained by following phenotypic leads.

Drug Repurposing for Influenza Virus Polymerase Acidic (PA) Endonuclease Inhibitor

Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PAN) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T’s active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.

Capillary electrophoresis-based functional genomics screening to discover novel archaeal DNA modifying enzymes

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis ( T. kodakarensis ). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli . RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

A simple and general approach to control the activity of DNA processing enzymes

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays, and showed comparable performance to commercial hot-start enzymes.

EXTENDED TUDOR-DOMAINS of the piRNA BIOGENESIS PATHWAY HAVE RNA-SPECIFIC NUCLEASE ACTIVITY

piRNAs are essential for transposon repression and protecting the germline from deleterious mutations. piRNA biogenesis comprises a primary and secondary pathway, and involves PIWI clade argonaute proteins and ancillary factors. Secondary piRNA biogenesis is tightly coupled to transposon repression. It requires processing of the 3-prime end of pre-piRNA during an amplification loop by an as yet unidentified endonuclease. Here, using crystallography, and biochemical assays, we discover that the Drosophila Qin protein, which is a critical member of the core amplification complex, has endonuclease activity. Qin contains five extended Tudor domains, which had been proposed to recognize methylated ligands. Instead, we show that these domains act as RNA-specific nucleases. This supports a role for Qin in the 3-prime end processing of Ago3-bound pre-piRNAs. Extended Tudor domains are frequent in piRNA-processing proteins, suggesting that the uncovered nuclease activity of this protein fold may be key to understanding the piRNA biogenesis.

Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions; cryo-EM structures of collided disomes from E. coli and B. subtilis reveal a distinct and conserved arrangement of the individual ribosomes and the composite SmrB binding site. These findings reveal the underlying mechanism by which ribosome collisions trigger ribosome rescue in bacteria.