DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites

The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in Streptomyces lividans 1326, was strongly aggravated when one (dndB) of the five genes (dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid (pHZ209), reflecting DNA degradation, displayed a clear change from a preferential modification site in strain 1326 to more random modifications in the mutant. Fourteen randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be modified only once. Residues in a region (5′-c–cGGCCgccg-3′) including a highly conserved 4-bp central core (5′-GGCC-3′) in a well-documented preferential modification site were assessed for their necessity by site-directed mutagenesis. While the central core (GGCC) was found to be stringently required in 1326 and in the mutant, ‘gccg’ flanking its right could either abolish or reduce the modification frequency only in the mutant, and two separate nucleotides to the left had no dramatic effect. The lack of essentiality of DndB for S-modification suggests that it might only be required for enhancing or stabilizing the activity of a protein complex at the required preferential modification site, or resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle for efficient modification

Regulation of dndB Gene Expression in Streptomyces lividans

DNA sulfur modification is a unique modification occurring in the sugar-phosphate backbone of DNA, with a nonbridging oxygen atom substituted with sulfur in a sequence-specific and Rp stereo-specific manner. Bioinformatics, RNA-seq, and in vitro transcriptional analyses have shown that DNA sulfur modification may be involved in epigenetic regulation. However, the in vivo evidence supporting this assertion is not convincing. Here, we aimed to characterize two sulfur-modified sites near the dndB promoter region in Streptomyces lividans. Single mutation of either site had no effect on dndB transcription, whereas double mutation of both sites significantly elevated dndB expression. These findings suggested that DNA sulfur modification affected gene expression, and the role of DNA sulfur modification in epigenetic regulation depended on the number of sulfur-modified sites. We also identified an inverted repeat, the R repeat sequence, and showed that this sequence participated in the positive regulation of dndB gene expression

Coded-aperture broadband light field imaging using digital micromirror devices

Coded-aperture light field (CALF) imaging can record four-dimensional information of incident light rays with a high angular resolution while retaining a camera's full pixel count. However, existing systems are limited by either imaging speeds and image contrasts of liquid-crystal spatial light modulators or by severe dispersion to broadband light of digital micromirror devices (DMDs), both of which hinder CALF's widespread applications. Here, we overcome these limitations by developing dispersion-eliminated (DE) CALF imaging. Using a dual-DMD design to compensate for dispersion in the entire visible spectrum, the DECALF imaging system captures 1280 x 1024 x 5 x 5 (x, y, theta, phi) color light field images at 20 Hz. Using static and dynamic three-dimensional (3D) color scenes, we experimentally demonstrate multi-perspective viewing, digital refocusing, and 3D tracking of the DECALF imaging system. We also apply it to the imaging and analyses of escape behaviors of freely moving normal and disease-model zebrafish. (C) 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique "H"-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and L-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents

Crystal Structure of the Cysteine Desulfurase DndA from Streptomyces lividans Which Is Involved in DNA Phosphorothioation

DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA

Does Involution Cause Anxiety? An Empirical Study from Chinese Universities

The debate over whether involution causes anxiety has persisted because no studies have attempted to quantify introversion and study its relationship to anxiety. This study quantified involution and explored its relationship with anxiety, provided evidence about whether involution was related to anxiety, and created a foundation for other scholars to carry out research on involution. Interviews and questionnaires were conducted to investigate the characteristics of 535 Chinese college students’ involution behavior and its relationship with anxiety. We found that involution was not necessarily positively related to anxiety. The specific results were as follows: (1) The involution behavior of the Chinese college students could be divided into three types: the passive involution, reward-oriented involution, and achievement-motivated involution; (2) Significant differences in the involvement of involution existed at the college level; (3) Three motivations that resulted in involution, from primary to secondary, were achievement-motivation, reward-orientation, and passive engagement; and (4) Passive involution, reward-oriented involution, and the total scores for the involution behavior of the college students were significantly and positively correlated with anxiety. Among the three types of involution behavior, the college students’ passive involution had a significant and positive predictive effect on their anxiety, while achievement-motivated involution had a significant and negative predictive effect

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882▿

The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel

High-precision 3D printing of high-strength polymer-derived ceramics: impact of precursor's molecular structure

Additive manufacturing (3D printing) offers new opportunities to create complex structures for many applications. The development of suitable precursors for high-resolution 3D printing of ceramics is increasingly essential to meet evolving material requirements. Herein, a new hybrid preceramic formulation based on thiol-ene click chemistry for precision printing of polymer-derived ceramic has been developed to enable the fabrication of complex 3D objects using high-resolution projection microstereolithography (PμSL). Two low-odor thiol compounds with either three (trithiol) or four thiols (tetrathiol) moieties have been examined, to investigate the influence of thiol structure on the mechanical properties of converted ceramics. Pyrolysis of the printed green bodies leads to the formation of silicon oxycarbide (SiOC) with high fidelity after polymer-to-ceramic transformation. The SiOC printed specimen converted from the tetrathiol formulation (4T) demonstrates excellent mechanical strength surpassing that of the trithiol-based formulation (3T) and previously reported SiOC preceramic polymers. The ceramic honeycomb fabricated using the tetrathiol compound shows remarkable improvement in compressive strength, which is two times higher than that of the trithiol-derived ceramic. This work proposes a simple and effective way to formulate 3D printable preceramic polymers through molecular design. The achieved 3D printed SiOC can fulfill the requirement for high-strength ceramic materials with complex shapes.Nanyang Technological UniversityThe authors acknowledge support from the Nanyang Technological University (NTU) with grant number of 04IDS000677N040