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.

Condensin-Mediated Chromosome Folding and Internal Telomeres Drive Dicentric Severing by Cytokinesis.

In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.

Sir3 heterochromatin protein promotes non-homologous end joining by direct inhibition of Sae2.

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.

Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps.

The bypass of DNA lesions that block replicative polymerases during DNA replication relies on DNA damage tolerance pathways. The error-prone translesion synthesis (TLS) pathway depends on specialized DNA polymerases that incorporate nucleotides in front of base lesions, potentially inducing mutagenesis. Two error-free pathways can bypass the lesions: the template switching pathway, which uses the sister chromatid as a template, and the homologous recombination pathway (HR), which also can use the homologous chromosome as template. The balance between error-prone and error-free pathways controls the mutagenesis level. Therefore, it is crucial to precisely characterize factors that influence the pathway choice to better understand genetic stability at replication forks. In yeast, the complex formed by the Rad51 paralogs Rad55 and Rad57 promotes HR and template-switching at stalled replication forks. At DNA double-strand breaks (DSBs), this complex promotes Rad51 filament formation and stability, notably by counteracting the Srs2 anti-recombinase. To explore the role of the Rad55-Rad57 complex in error-free pathways, we monitored the genetic interactions between Rad55-Rad57, the translesion polymerases Polζ or Polη, and Srs2 following UV radiation that induces mostly single-strand DNA gaps. We found that the Rad55-Rad57 complex was involved in three ways. First, it protects Rad51 filaments from Srs2, as it does at DSBs. Second, it promotes Rad51 filament stability independently of Srs2. Finally, we observed that UV-induced HR is almost abolished in Rad55-Rad57 deficient cells, and is partially restored upon Polζ or Polη depletion. Hence, we propose that the Rad55-Rad57 complex is essential to promote Rad51 filament stability on single-strand DNA gaps, notably to counteract the error-prone TLS polymerases and mutagenesis.

Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability.

In cells lacking telomerase, telomeres gradually shorten during each cell division to reach a critically short length, permanently activate the DNA damage checkpoint, and trigger replicative senescence. The increase in genome instability that occurs as a consequence may contribute to the early steps of tumorigenesis. However, because of the low frequency of mutations and the heterogeneity of telomere-induced senescence, the timing and mechanisms of genome instability increase remain elusive. Here, to capture early mutation events during replicative senescence, we used a combined microfluidic-based approach and live-cell imaging in yeast. We analyzed DNA damage checkpoint activation in consecutive cell divisions of individual cell lineages in telomerase-negative yeast cells and observed that prolonged checkpoint arrests occurred frequently in telomerase-negative lineages. Cells relied on the adaptation to the DNA damage pathway to bypass the prolonged checkpoint arrests, allowing further cell divisions despite the presence of unrepaired DNA damage. We demonstrate that the adaptation pathway is a major contributor to the genome instability induced during replicative senescence. Therefore, adaptation plays a critical role in shaping the dynamics of genome instability during replicative senescence.

Cytokinesis breaks dicentric chromosomes preferentially at pericentromeric regions and telomere fusions.

Dicentric chromosomes are unstable products of erroneous DNA repair events that can lead to further genome rearrangements and extended gene copy number variations. During mitosis, they form anaphase bridges, resulting in chromosome breakage by an unknown mechanism. In budding yeast, dicentrics generated by telomere fusion break at the fusion, a process that restores the parental karyotype and protects cells from rare accidental telomere fusion. Here, we observed that dicentrics lacking telomere fusion preferentially break within a 25- to 30-kb-long region next to the centromeres. In all cases, dicentric breakage requires anaphase exit, ruling out stretching by the elongated mitotic spindle as the cause of breakage. Instead, breakage requires cytokinesis. In the presence of dicentrics, the cytokinetic septa pinch the nucleus, suggesting that dicentrics are severed after actomyosin ring contraction. At this time, centromeres and spindle pole bodies relocate to the bud neck, explaining how cytokinesis can sever dicentrics near centromeres.

Recombination at subtelomeres is regulated by physical distance, double-strand break resection and chromatin status.

Homologous recombination (HR) is a conserved mechanism that repairs broken chromosomes via intact homologous sequences. How different genomic, chromatin and subnuclear contexts influence HR efficiency and outcome is poorly understood. We developed an assay to assess HR outcome by gene conversion (GC) and break-induced replication (BIR), and discovered that subtelomeric double-stranded breaks (DSBs) are preferentially repaired by BIR despite the presence of flanking homologous sequences. Overexpression of a silencing-deficient SIR3 mutant led to active grouping of telomeres and specifically increased the GC efficiency between subtelomeres. Thus, physical distance limits GC at subtelomeres. However, the repair efficiency between reciprocal intrachromosomal and subtelomeric sequences varies up to 15-fold, depending on the location of the DSB, indicating that spatial proximity is not the only limiting factor for HR EXO1 deletion limited the resection at subtelomeric DSBs and improved GC efficiency. The presence of repressive chromatin at subtelomeric DSBs also favoured recombination, by counteracting EXO1-mediated resection. Thus, repressive chromatin promotes HR at subtelomeric DSBs by limiting DSB resection and protecting against genetic information loss.

Keep moving and stay in a good shape to find your homologous recombination partner.

Genomic DNA is constantly exposed to damage. Among the lesion in DNA, double-strand breaks (DSB), because they disrupt the two strands of the DNA double helix, are the more dangerous. DSB are repaired through two evolutionary conserved mechanisms: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). Whereas NHEJ simply reseals the double helix with no or minimal processing, HR necessitates the formation of a 3'ssDNA through the processing of DSB ends by the resection machinery and relies on the recognition and pairing of this 3'ssDNA tails with an intact homologous sequence. Despite years of active research on HR, the manner by which the two homologous sequences find each other in the crowded nucleus, and how this modulates HR efficiency, only recently emerges. Here, we review recent advances in our understanding of the factors limiting the search of a homologous sequence during HR.

Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions.

Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes or nuclear bodies, but the resulting forces cannot be directly measured in vivo. However, they impact chromatin dynamics and should be reflected in particular in the motion of a single locus. We present here a method based on polymer models and statistics of single trajectories to extract the force characteristics and in particular when they are generated by the gradient of a quadratic potential well. Using numerical simulations of a Rouse polymer and live cell imaging of the MAT-locus located on the yeast Saccharomyces cerevisiae chromosome III, we recover the amplitude and the distance between the observed and the interacting monomer. To conclude, the confined trajectories we observed in vivo reflect local interaction on chromatin.

On the edge: how nuclear pore complexes rule genome stability.

Nuclear organization has emerged as a critical layer in the coordination of DNA repair activities. Distinct types of DNA lesions have notably been shown to relocate at the vicinity of nuclear pore complexes (NPCs), where specific repair pathways are favored, ultimately safeguarding genome integrity. Here, we review the most recent progress in this field, notably highlighting the increasingly diverse types of DNA structures undergoing repositioning, and the signaling pathways involved. We further discuss our growing knowledge of the molecular mechanisms underlying the choice of repair pathways at NPCs, and their conservation - or divergences. Intriguingly, a series of recent findings suggest that DNA metabolism may be coupled to NPC biogenesis and specialization, challenging our initial vision of these processes.

A R-loop sensing pathway mediates the relocation of transcribed genes to nuclear pore complexes.

Nuclear pore complexes (NPCs) have increasingly recognized interactions with the genome, as exemplified in yeast, where they bind transcribed or damaged chromatin. By combining genome-wide approaches with live imaging of model loci, we uncover a correlation between NPC association and the accumulation of R-loops, which are genotoxic structures formed through hybridization of nascent RNAs with their DNA templates. Manipulating hybrid formation demonstrates that R-loop accumulation per se, rather than transcription or R-loop-dependent damages, is the primary trigger for relocation to NPCs. Mechanistically, R-loop-dependent repositioning involves their recognition by the ssDNA-binding protein RPA, and SUMO-dependent interactions with NPC-associated factors. Preventing R-loop-dependent relocation leads to lethality in hybrid-accumulating conditions, while NPC tethering of a model hybrid-prone locus attenuates R-loop-dependent genetic instability. Remarkably, this relocation pathway involves molecular factors similar to those required for the association of stalled replication forks with NPCs, supporting the existence of convergent mechanisms for sensing transcriptional and genotoxic stresses.

Distance between homologous chromosomes results from chromosome positioning constraints.

The organization of chromosomes is important for various biological processes and is involved in the formation of rearrangements often observed in cancer. In mammals, chromosomes are organized in territories that are radially positioned in the nucleus. However, it remains unclear whether chromosomes are organized relative to each other. Here, we examine the nuclear arrangement of 10 chromosomes in human epithelial cancer cells by three-dimensional FISH analysis. We show that their radial position correlates with the ratio of their gene density to chromosome size. We also observe that inter-homologue distances are generally larger than inter-heterologue distances. Using numerical simulations taking radial position constraints into account, we demonstrate that, for some chromosomes, radial position is enough to justify the inter-homologue distance, whereas for others additional constraints are involved. Among these constraints, we propose that nucleolar organizer regions participate in the internal positioning of the acrocentric chromosome HSA21, possibly through interactions with nucleoli. Maintaining distance between homologous chromosomes in human cells could participate in regulating genome stability and gene expression, both mechanisms that are key players in tumorigenesis.

DNA Repair in Space and Time: Safeguarding the Genome with the Cohesin Complex.

DNA double-strand breaks (DSBs) are a deleterious form of DNA damage, which must be robustly addressed to ensure genome stability. Defective repair can result in chromosome loss, point mutations, loss of heterozygosity or chromosomal rearrangements, which could lead to oncogenesis or cell death. We explore the requirements for the successful repair of DNA DSBs by non-homologous end joining and homology-directed repair (HDR) mechanisms in relation to genome folding and dynamics. On the occurrence of a DSB, local and global chromatin composition and dynamics, as well as 3D genome organization and break localization within the nuclear space, influence how repair proceeds. The cohesin complex is increasingly implicated as a key regulator of the genome, influencing chromatin composition and dynamics, and crucially genome organization through folding chromosomes by an active loop extrusion mechanism, and maintaining sister chromatid cohesion. Here, we consider how this complex is now emerging as a key player in the DNA damage response, influencing repair pathway choice and efficiency.

Degradation of S. cerevisiae Cohesin with the Auxin-Inducible Degron System.

The cohesin complex is required to establish sister chromatid cohesion and ensure accurate chromosome segregation after DNA replication. Recent data has also revealed a role for cohesion as a major player in DNA repair and gene expression regulation. All subunits of the Cohesin complex are essential and cannot be deleted. Here, we describe a protocol to efficiently deplete cohesin subunits with an auxin-inducible degron (AID) system in S. cerevisiae.

Nej1 Interacts with Mre11 to Regulate Tethering and Dna2 Binding at DNA Double-Strand Breaks.

Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end-tethering at a DSB, and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to the break. Nej1 and Dna2-Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. By characterizing the functionality of Nej1 in two rad50 mutants, which alter the structural features of MRX, we demonstrate that Nej1 inhibits the binding of Dna2 to Mre11 and Sgs1. Nej1 interactions with Mre11 promote tethering and inhibit hyper-resection, and when these events are compromised, large deletions develop at a DSB. The work indicates that Nej1 provides a layer of regulation to repair pathway choice and is consistent with its role in NHEJ.

Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast.

BACKGROUND: The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle. RESULTS: We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms. CONCLUSIONS: Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Telomeres and telomerase as targets for anticancer drug development.

In most human cancers, the telomere erosion problem has been bypassed through the activation of a telomere maintenance system (usually activation of telomerase). Therefore, telomere and telomerase are attractive targets for anti-cancer therapeutic interventions. Here, we review a large panel of strategies that have been explored to date, from small inhibitors of the catalytic sub-unit of telomerase to anti-telomerase immunotherapy and gene therapy. The many positive results that are reported from anti-telomere/telomerase assays suggest a prudent optimism for a possible clinical application in a close future. However, we discuss some of the main limits for these approaches of antitumour drug development and why significant work remains before a clinically useful drug can be proposed to patients.

Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase.

Recent findings suggest important roles for nuclear organization in gene expression. In contrast, little is known about how nuclear organization contributes to genome stability. Epistasis analysis (E-MAP) using DNA repair factors in yeast indicated a functional relationship between a nuclear pore subcomplex and Slx5/Slx8, a small ubiquitin-like modifier (SUMO)-dependent ubiquitin ligase, which we show physically interact. Real-time imaging and chromatin immunoprecipitation confirmed stable recruitment of damaged DNA to nuclear pores. Relocation required the Nup84 complex and Mec1/Tel1 kinases. Spontaneous gene conversion can be enhanced in a Slx8- and Nup84-dependent manner by tethering donor sites at the nuclear periphery. This suggests that strand breaks are shunted to nuclear pores for a repair pathway controlled by a conserved SUMO-dependent E3 ligase.

The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast.

Double-strand breaks (DSB) in yeast lead to the formation of repair foci and induce a checkpoint response that requires both the ATR-related kinase Mec1 and its target, Rad53. By combining high-resolution confocal microscopy and chromatin-immunoprecipitation assays, we analysed the genetic requirements for and the kinetics of Mec1 recruitment to an irreparable HO-endonuclease-induced DSB. Coincident with the formation of a 3' overhang, the Mec1-Ddc2 (Lcd1) complex is recruited into a single focus that colocalises with the DSB site and precipitates with single-strand DNA (ssDNA). The absence of Rad24 impaired cut-site resection, Mec1 recruitment and focus formation, whereas, in the absence of yKu70, both ssDNA accumulation and Mec1 recruitment was accelerated. By contrast, mutation of the N-terminus of the large RPA subunit blocked Mec1 focus formation without affecting DSB processing, arguing for a direct involvement of RPA in Mec1-Ddc2 recruitment. Conversely, loss of Rad51 enhanced Mec1 focus formation independently of ssDNA formation, suggesting that Rad51 might compete for the interaction of RPA with Mec1-Ddc2. In all cases, Mec1 focus formation correlated with checkpoint activation. These observations led to a model that links end-processing and competition between different ssDNA-binding factors with Mec1-Ddc2 focus formation and checkpoint activation.