Multi-invasions Are Recombination Byproducts that Induce Chromosomal Rearrangements.

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3'-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2, and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.

Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae.

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization.

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability.

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

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.

Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

Multi-Invasion-Induced Rearrangements as a Pathway for Physiological and Pathological Recombination.

Cells mitigate the detrimental consequences of DNA damage on genome stability by attempting high fidelity repair. Homologous recombination templates DNA double-strand break (DSB) repair on an identical or near identical donor sequence in a process that can in principle access the entire genome. Other physiological processes, such as homolog recognition and pairing during meiosis, also harness the HR machinery using programmed DSBs to physically link homologs and generate crossovers. A consequence of the homology search process by a long nucleoprotein filament is the formation of multi-invasions (MI), a joint molecule in which the damaged ssDNA has invaded more than one donor molecule. Processing of MI joint molecules can compromise the integrity of both donor sites and lead to their rearrangement. Here, two mechanisms for the generation of rearrangements as a pathological consequence of MI processing are detailed and the potential relevance for non-allelic homologous recombination discussed. Finally, it is proposed that MI-induced crossover formation may be a feature of physiological recombination.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites.

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo.

Moving forward one step back at a time: reversibility during homologous recombination.

DNA double-strand breaks are genotoxic lesions whose repair can be templated off an intact DNA duplex through the conserved homologous recombination (HR) pathway. Because it mainly consists of a succession of non-covalent associations of molecules, HR is intrinsically reversible. Reversibility serves as an integral property of HR, exploited and tuned at various stages throughout the pathway with anti- and pro-recombinogenic consequences. Here, we focus on the reversibility of displacement loops (D-loops), a central DNA joint molecule intermediate whose dynamics and regulation have recently been physically probed in somatic S. cerevisiae cells. From homology search to repair completion, we discuss putative roles of D-loop reversibility in repair fidelity and outcome.

Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C.

In chromosome conformation capture experiments (Hi-C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn-HiC design). In the Syn-HiC region, Hi-C signal-to-noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies, allowing to propose a robust definition of a Hi-C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene-arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8-delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions.

G-quadruplex-induced instability during leading-strand replication.

G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.

Stimulation of gross chromosomal rearrangements by the human CEB1 and CEB25 minisatellites in Saccharomyces cerevisiae depends on G-quadruplexes or Cdc13.

Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC-rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC(3) G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites' distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability.

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide sequencing approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis, and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR sub-pathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology, and results in sequence insertion without additional break or SV. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants.

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae.

G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.

The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Recombination-mediated genome rearrangements.

Homologous recombination (HR) is a universal DNA double-strand break (DSB) repair pathway that uses an intact DNA molecule as a template. Signature HR reactions are homology search and DNA strand invasion catalyzed by the prototypical RecA-ssDNA filament (Rad51 and Dmc1 in eukaryotes), which produces heteroduplex DNA-containing joint molecules (JMs). These reactions uniquely infringe on the DNA strands association established at replication, on the basis of substantial sequence similarity. For that reason, and despite the high fidelity of its templated nature, DSB repair by HR authorizes the alteration of genome structure, guided by repetitive DNA elements. The resulting structural variations (SVs) can involve vast genomic regions, potentially affecting multiple coding sequences and regulatory elements at once, with possible pathological consequences. Here, we discuss recent advances in our understanding of genetic and molecular vulnerabilities of HR leading to SVs, and of the various fidelity-enforcing factors acting across scales on the balancing act of this complex pathway. An emphasis is put on extra-chomosomal DNAs, both product of, and substrate for HR-mediated chromosomal rearrangements.

Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae.

DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.

Cohesin regulates homology search during recombinational DNA repair.

Homologous recombination repairs DNA double-strand breaks (DSB) using an intact dsDNA molecule as a template. It entails a homology search step, carried out along a conserved RecA/Rad51-ssDNA filament assembled on each DSB end. Whether, how and to what extent a DSB impacts chromatin folding, and how this (re)organization in turns influences the homology search process, remain ill-defined. Here we characterize two layers of spatial chromatin reorganization following DSB formation in Saccharomyces cerevisiae. Although cohesin folds chromosomes into cohesive arrays of ~20-kb-long chromatin loops as cells arrest in G2/M, the DSB-flanking regions interact locally in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin, Mec1ATR, Rad52 and Rad51. This local structure blocks cohesin progression, constraining the DSB region at the base of a loop. Functionally, cohesin promotes DSB-dsDNA interactions and donor identification in cis, while inhibiting them in trans. This study identifies multiple direct and indirect ways by which cohesin regulates homology search during recombinational DNA repair.

Rdh54/Tid1 inhibits Rad51-Rad54-mediated D-loop formation and limits D-loop length.

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.