Impact of R-loops on oncogene-induced replication stress in cancer cells.

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.

Le stress réplicatif correspond à une altération de la progression des fourches de réplication causé par une variété d'événements d'origine endogène ou exogène. Dans les lésions précancéreuses, ce stress est aggravé par la dérégulation de voies oncogéniques, qui perturbe notamment la coordination entre la réplication et la transcription du génome et entraine une instabilité génétique contribuant au développement du cancer. Il est maintenant bien établi que la transcription peut interférer avec la réplication du génome de différentes façons, telles que des collisions frontales entre polymérases, l'accumulation de supertours positifs de l'ADN ou la formation de R-loops. Ces structures se forment au cours de la transcription lorsque l'ARN naissant se réassocie avec l'ADN derrière l'ARN polymérase, formant un hybride ADN :ARN stable. Dans cette revue, nous discutons comment ces différents processus cotranscriptionnels perturbent la progression des fourches de réplication et comment ils contribuent à l'instabilité génétique des cellules cancéreuses.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart.

R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

Sen1 is a key regulator of transcription-driven conflicts.

Cellular homeostasis requires the coordination of several machineries concurrently engaged in the DNA. Wide-spread transcription can interfere with other processes, and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase not only terminates non-coding transcription but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability, but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that, under physiological conditions, Sen1 removes RNA polymerase II at TRCs within genes and the rDNA and at sites of transcription-transcription conflicts, thus qualifying as a "key regulator of conflicts." We demonstrate that genomic stability is affected by Sen1 mutation only when in addition to its role at the replisome, the termination of non-coding transcription or R-loop removal are additionally compromised.

Toxic R-loops: Cause or consequence of replication stress?

Transcription-replication conflicts (TRCs) represent a potential source of endogenous replication stress (RS) and genomic instability in eukaryotic cells but the mechanisms that underlie this instability remain poorly understood. Part of the problem could come from non-B DNA structures called R-loops, which are formed of a RNA:DNA hybrid and a displaced ssDNA loop. In this review, we discuss different scenarios in which R-loops directly or indirectly interfere with DNA replication. We also present other types of TRCs that may not depend on R-loops to impede fork progression. Finally, we discuss alternative models in which toxic RNA:DNA hybrids form at stalled forks as a consequence - but not a cause - of replication stress and interfere with replication resumption.

3D positioning of tagged DNA loci by widefield and super-resolution fluorescence imaging of fixed yeast nuclei.

This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment. For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.

The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.

Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing.

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.

Dbf4 recruitment by forkhead transcription factors defines an upstream rate-limiting step in determining origin firing timing.

Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, dbf4ΔC, phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.

Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome.

DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1's role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.

Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance.

The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.

Closing the MCM cycle at replication termination sites.

The initiation of eukaryotic DNA replication is a highly regulated process conserved from yeast to human. The past decade has seen significant advances in understanding how the CMG (Cdc45­MCM­GINS) replicative helicase is loaded onto DNA. However, very little was known on how this complex is removed from chromatin at the end of S phase. Two papers in a recent issue of Science [1], [2] show that in yeast and in Xenopus, the CMG complex is unloaded at replication termination sites by an active mechanism involving the polyubiquitylation of Mcm7.

The histone deacetylases sir2 and rpd3 act on ribosomal DNA to control the replication program in budding yeast.

In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3Δ cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors.

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression.

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.

The replication timing program in the hands of two HDACs.

In eukaryotes, duplication of genomic information depends on the sequential activation of multiple replication origins distributed along the chromosomes. Replication origins differ in initiation time, chromatin structure and three-dimensional position in the nucleus. Recently, we have performed a systematic analysis of the role of histone deacetylases (HDACs) in the regulation of origin activity in budding yeast. We have found that the epigenetic regulation of repetitive sequences is a key determinant of the DNA replication program. Indeed, our study revealed that two histone deacetylases, Rpd3 and Sir2, have opposite effects on the replication timing program. Rpd3 delays initiation at late origins, whereas Sir2 promotes efficient activation of early origins. Remarkably, we also found that Rpd3 and Sir2 regulate initiation at ~200 replication origins located within the ribosomal DNA (rDNA) array. We propose that this epigenetic regulation of repetitive origins controls the replication timing program by modulating the availability of limiting initiation factors.

Budding yeast Wapl controls sister chromatid cohesion maintenance and chromosome condensation.

The establishment of stable sister chromatid cohesion during DNA replication requires acetylation of the chromosomal cohesin complex by the replication fork-associated acetyltransferase Eco1. Cohesin acetylation is thought to facilitate replication fork progression by counteracting an as yet ill-defined cohesion "antiestablishment" activity imposed by the Wapl protein. Here, using budding yeast, we find no evidence that cohesin acetylation must overcome Wapl during replication fork progression. Instead, Wapl emerges as a negative regulator of cohesion maintenance in G2, a function that it likely exerts through its role as destabilizer of unacetylated, chromosome-bound cohesin. Our results suggest that acetylation renders cohesin Wapl-resistant from S phase onward until mitosis. In the absence of Wapl, sister chromatid cohesion functions well, suggesting that Wapl partakes in a cohesin function outside of sister chromatid cohesion. We find that Wapl is not required for cohesin's known role in transcriptional regulation. Rather, cells lacking Wapl display increased chromosome condensation in both interphase and mitosis. Thus, as a conserved regulator of cohesin dynamics on chromosomes, Wapl controls cohesion maintenance after its establishment in S phase and adjusts the chromosome condensation status.

Cohesin association to replication sites depends on rad50 and promotes fork restart.

The cohesin complex holds together newly replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins, and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is critical for the recovery of stalled forks. Cohesin enrichment at replication forks does not depend on γH2A(X) formation, which differs from its loading requirements at DNA double-strand breaks (DSBs). However, cohesin localization is largely reduced in rad50Δ mutants and in cells lacking both Mec1 and Tel1 checkpoint kinases. Interestingly, cohesin loading at replication sites depends on the structural features of Rad50 that are important for bridging sister chromatids, including the CXXC hook domain and the length of the coiled-coil extensions. Together, these data reveal a function for cohesin in the maintenance of genome integrity during S phase.