The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements (MGEs) encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all characterized Acrs directly bind Cas proteins to inactivate CRISPR immunity. Here, using functional metagenomic selection, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of clostridial bacteria and their prophages from human gut microbiomes. AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic acid–binding proteins. Based on mutational analyses and functional assays, we deduced that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids. This may render them less susceptible to SpyCas9, which uses free energy from negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and MGEs.

AcrIIA22 is a novel anti-CRISPR that impairs SpyCas9 activity by relieving DNA torsion of target plasmids

To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all mechanistically characterized Acrs directly bind their cognate Cas protein to inactivate CRISPR immunity. Here, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of Clostridial bacteria and their prophages from the human gut microbiome. Uncovered in a functional metagenomic selection, AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. This homology helped us deduce that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids, rendering them less susceptible to SpyCas9, which is highly dependent on negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

The Stringent Response Inhibits DNA Replication Initiation inE. coliby Modulating Supercoiling oforiC

ABSTRACTThe stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation inEscherichia coliremains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factordnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication,oriC. Active transcription normally introduces negative supercoils intooriCto help promote replication initiation, so the accumulation of ppGpp reduces initiation potential atoriCby reducing transcription. We find that maintaining transcription nearoriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein geneseqAalso relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils intooriC.IMPORTANCETo survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation inEscherichia colifor decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, as well as evolution across species. Head-on conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts (leading strand genes). The source of this fundamental difference is unknown. Here, we report that topological stress underlies this difference. We find that head-on conflict resolution requires the relaxation of positive supercoils DNA gyrase and Topo IV. Interestingly, we find that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through gyrase activity is most likely caused by the diffusion of negative supercoils through RNA polymerase spinning. Altogether, our results address a longstanding question regarding replication-transcription conflicts by revealing the fundamental mechanistic difference between the two types of encounters.

Supercoiling Theory and Model of Chromosomal Structures in Eukaryotic Cells

About six billion base pairs of DNA reside highly orderly in each human cell’s nucleus through their manifestation as twenty-three pairs of chromosomes. Delicate patterns of spatial organizations of DNA macromolecules in these eukaryotic chromosomes as well as their associated physical driving forces have, however, not been fully understood thus far. On the basis of (1) our four recent discoveries about supercoiling properties of histone H1, nucleosomes, linker DNA and polynucleosomes, (2) well-accepted six axioms about signs, shapes and handedness of DNA supercoils, and (3) our three new prepositions about correlations between DNA supercoils and chromosomal structures, we formulate new theories and models of eukaryotic chromosomal structures in the current report. It is our conclusion that all levels of chromosomal structures in eukaryotic cells are governed mainly by negative supercoils that are present in their naked linker DNA regions.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35–38 Amino Acids of GyrA

Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.

Topoisomerases

This chapter discusses single-molecule approaches in the study of topoisomerases. After introducing the problem posed by DNA entanglement, it describes type I and type II topoisomerases, which solve that issue. Single-molecule assays have nailed down the different mechanisms of bacterial and eukaryotic type I topoisomerases. The properties of type II topoisomerases are then described. Single-molecule experiments have shown that they relax DNA torsion by two units, passing one dsDNA segment through a break in another segment. However, while topoII relaxes positive and negative supercoils similarly, topoIV relaxes positive supercoils quickly and processively, but negative ones slowly and distributively. This chiral discrimination is compared with the activity of the enzyme on two catenated DNA molecules. After describing single-molecule assays of the activity of gyrases, in-vivo investigations of single fluorescently labelled topoIV units in single E.coli are discussed, with concluding remarks on the future of single-molecule DNA/protein studies.

Supercoiling Theory and Model of Chromosomal Structures in Eukaryotic Cells

About six billion base pairs of DNA reside highly orderly in each human cell’s nucleus through their manifestation as twenty-three pairs of chromosomes. Delicate patterns of spatial organizations of DNA macromolecules in these eukaryotic chromosomes as well as their associated physical driving forces have, however, not been fully understood thus far. On the basis of (1) our four recent discoveries about supercoiling properties of histone H1, nucleosomes, linker DNA and polynucleosomes and (2) well-established axioms about signs, shapes and handedness of DNA supercoils, we formulate new theories and models of eukaryotic chromosomal structures. It is our conclusion that three-dimensional structures of eukaryotic chromosomes and their sublevel architectures are govern mainly by negative supercoils that are present in their naked linker DNA regions.

DNA Topoisomerase I differentially modulates R-loops across the human genome

ABSTRACTBackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.ResultsHere, we performed high-resolution strand-specific R-loop mapping in human cells depleted for Top 1 and found that Top1 depletion resulted in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains were characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occurred in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincided with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.ConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis and suggest a potential role for Top1 in controlling replication initiation via R-loop formation.

Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

ABSTRACTDNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind bothEscherichia coliandMycobacterium tuberculosisgyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while inE. coliholoenzyme is the target. The binding of MFX to GyrA ofM. tuberculosiscorrelates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.