Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

Fork pausing complex engages topoisomerases at the replisome.

Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) act as a "molecular brake" and promote fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that the Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a "sTOP" mechanism ("slowing down with topoisomerases I-II"), which we show also contributes to protecting cells from topoisomerase-blocking agents.

Dynamic Processing of Displacement Loops during Recombinational DNA Repair.

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.

Biochemical characterization of the meiosis-essential yet evolutionarily divergent topoisomerase VIB-like protein MTOPVIB from Arabidopsis thaliana.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.

Nucleases Acting at Stalled Forks: How to Reboot the Replication Program with a Few Shortcuts.

In a lifetime, a human being synthesizes approximately 2×1016 meters of DNA, a distance that corresponds to 130,000 times the distance between the Earth and the Sun. This daunting task is executed by thousands of replication forks, which progress along the chromosomes and frequently stall when they encounter DNA lesions, unusual DNA structures, RNA polymerases, or tightly-bound protein complexes. To complete DNA synthesis before the onset of mitosis, eukaryotic cells have evolved complex mechanisms to process and restart arrested forks through the coordinated action of multiple nucleases, topoisomerases, and helicases. In this review, we discuss recent advances in understanding the role and regulation of nucleases acting at stalled forks with a focus on the nucleolytic degradation of nascent DNA, a process commonly referred to as fork resection. We also discuss the effects of deregulated fork resection on genomic instability and on the unscheduled activation of the interferon response under replication stress conditions.

The repair of topoisomerase 2 cleavage complexes in Arabidopsis.

DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.

A dual-activity topoisomerase complex promotes both transcriptional activation and repression in response to starvation.

Topoisomerases are required to release topological stress generated by RNA polymerase II (RNAPII) during transcription. Here, we show that in response to starvation, the complex of topoisomerase 3b (TOP3B) and TDRD3 can enhance not only transcriptional activation, but also repression, which mimics other topoisomerases that can also alter transcription in both directions. The genes enhanced by TOP3B-TDRD3 are enriched with long and highly-expressed ones, which are also preferentially stimulated by other topoisomerases, suggesting that different topoisomerases may recognize their targets through a similar mechanism. Specifically, human HCT116 cells individually inactivated for TOP3B, TDRD3 or TOP3B topoisomerase activity, exhibit similarly disrupted transcription for both starvation-activated genes (SAGs) and starvation-repressed genes (SRGs). Responding to starvation, both TOP3B-TDRD3 and the elongating form of RNAPII exhibit concomitantly increased binding to TOP3B-dependent SAGs, at binding sites that overlap. Notably, TOP3B inactivation decreases the binding of elongating RNAPII to TOP3B-dependent SAGs while increased it to SRGs. Furthermore, TOP3B-ablated cells display reduced transcription of several autophagy-associated genes and autophagy per se. Our data suggest that TOP3B-TDRD3 can promote both transcriptional activation and repression by regulating RNAPII distribution. In addition, the findings that it can facilitate autophagy may account for the shortened lifespan of Top3b-KO mice.

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.

Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.

Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis.

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5'/3' gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

DNA Topoisomerases in Unicellular Pathogens: Structure, Function, and Druggability.

All organisms, including unicellular pathogens, compulsorily possess DNA topoisomerases for successful nucleic acid metabolism. But particular subtypes of topoisomerases exist, in all prokaryotes and in some unicellular eukaryotes, that are absent in higher eukaryotes. Moreover, topoisomerases from pathogenic members of a niche possess some unique molecular architecture and functionalities completely distinct from their nonpathogenic colleagues. This review will highlight the unique attributes associated with the structures and functions of topoisomerases from the unicellular pathogens, with special reference to bacteria and protozoan parasites. It will also summarise the progress made in the domain pertaining to the druggability of these topoisomerases, upon which a future platform for therapeutic development can be successfully constructed.

All tangled up: how cells direct, manage and exploit topoisomerase function.

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.

Direct observation of helicase-topoisomerase coupling within reverse gyrase.

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.

What's on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases.

DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

Activation of multiple proteolysis systems contributes to acute cadmium cytotoxicity.

Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.

Frequent Interchromosomal Template Switches during Gene Conversion in S. cerevisiae.

Although repair of double-strand breaks (DSBs) by gene conversion is the most accurate way to repair such lesions, in budding yeast there is a 1,000-fold increase in accompanying mutations, including interchromosomal template switches (ICTS) involving highly mismatched (homeologous) ectopic sequences. Although such events are rare and appear at a rate of 2 × 10(-7) when template jumps occur between 71% identical sequences, they are surprisingly frequent (0.3% of all repair events) when the second template is identical to the first, revealing the remarkable instability of repair DNA synthesis. With homeologous donors, ICTS uses microhomologies as small as 2 bp. Cells lacking mismatch repair proteins Msh6 and Mlh1 form chimeric recombinants with two distinct patches of microhomology, implying that these proteins are crucial for strand discrimination of heteroduplex DNA formed during ICTS. We identify the chromatin remodeler Rdh54 as the first protein required for template switching that does not affect simple gene conversion.

SPO11.2 is essential for programmed double-strand break formation during meiosis in bread wheat (Triticum aestivum L.).

Meiotic recombination is initiated by formation of DNA double-strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11-1 and SPO11-2. We analysed the sequences of SPO11-2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11-2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon-2 of the A copy lead to a premature stop codon and suggest that it encodes a non-functional protein. Remarkably, the mutation was absent from the diploid A-relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11-2 in meiotic recombination in wheat. In accordance with its 2-nucleotide deletion in exon-2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus.

Maintaining an appropriate DNA topology with DNA-based processes (DNA replication, transcription and recombination) is crucial for all three domains of life. In bacteria, the homeostatic regulation for controlling DNA supercoiling relies on antagonistic activities of two DNA topoisomerases, TopoI and gyrase. In hyperthermophilic crenarchaea, the presence of such a regulatory system is suggested as two DNA topoisomerases, TopoVI and reverse gyrase, catalyze antagonistic activities. To test this hypothesis, we estimated and compared the number of the TopoVI with that of the two reverse gyrases, TopR1 and TopR2, in Sulfolobus solfataricus cells maintained either at 80 or at 88°C, or reciprocally shifted from one temperature to the other. From the three DNA topoisomerases, TopR1 is the only one exhibiting significant quantitative variations in response to the up- and down-shifts. In addition, the corresponding intrinsic activities of these three DNA topoisomerases were tested in vitro at both temperatures. Although temperature modulates the three DNA topoisomerases activities, TopR1 is the sole topoisomerase able to function at high temperature. Altogether, results presented in this study demonstrate, for the first time, that the DNA topological state of a crenarchaeon is regulated via a homeostatic control, which is mainly mediated by the fine-tuning of TopR1.

Topoisomerases and the regulation of neural function.

Topoisomerases are unique enzymes that regulate torsional stress in DNA to enable essential genome functions, including DNA replication and transcription. Although all cells in an organism require topoisomerases to maintain normal function, the nervous system in particular shows a vital need for these enzymes. Indeed, a range of inherited human neurologic syndromes, including neurodegeneration, schizophrenia and intellectual impairment, are associated with aberrant topoisomerase function. Much remains unknown regarding the tissue-specific function of neural topoisomerases or the connections between these enzymes and disease aetiology. Precisely how topoisomerases regulate genome dynamics within the nervous system is therefore a crucial research question.

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.