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Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes 1 . Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown 2 . Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes., (© 2023. The Author(s).)

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants - they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open 'O' shapes and collapsed 'B' shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.

To faithfully transmit genetic information, cells must replicate their entire genome before division. This is thought to be ensured by the temporal separation of replication and chromosome segregation. Here we show that in 20-40% of unperturbed yeast cells, DNA synthesis continues during anaphase, late in mitosis. High cyclin-Cdk activity inhibits DNA synthesis in metaphase, and the decrease in cyclin-Cdk activity during mitotic exit allows DNA synthesis to finish at subtelomeric and some difficult-to-replicate regions. DNA synthesis during late mitosis correlates with elevated mutation rates at subtelomeric regions, including copy number variation. Thus, yeast cells temporally overlap DNA synthesis and chromosome segregation during normal growth, possibly allowing cells to maximize population-level growth rate while simultaneously exploring greater genetic space.

DNA-strand breaks influence structure and function of chromosomes in diverse ways, and it is essential to analyze the lesions to understand behaviors of genetic information. For researchers in a wide array of fields including recombination, repair, and DNA damage response, efficient and easy detection of DNA breaks is of paramount importance. Among several procedures suitable for this purpose, a method to directly observe broken chromosomes by pulsed-field gel electrophoresis, using the fission yeast Schizosaccharomyces pombe as a model organism, is described in this chapter. Because S. pombe chromosomes are megabase-size, careful attention should be paid to maintain DNA as intact as possible. The protocol includes induction of DNA breaks, preparation of chromosomes, and separation of chromosomal DNA by PFGE. This procedure can be applicable to other species as well as other experiments handling large-size DNA molecules.

Separating DNA fragments using standard agarose gel electrophoresis is based on the capacity of negatively charged DNA molecules to move through the agarose gel matrix toward the positive electrode. Pulsed-field gel electrophoresis (PFGE) is an agarose gel electrophoresis technique that enables the separation of DNA molecules at a megabase scale, making the direct genomic analysis of large DNA molecules possible. For instance, 16 chromosomes (size range; 0.2-2.2 Mb) in Saccharomyces cerevisiae, whose karyotype cannot be easily observed with a microscope, can be directly separated on agarose gel. PFGE is also a powerful analytical tool for chromosomal mapping and genome structure analysis in bacterial and mammalian cells. In this chapter, we will describe the preparation of intact yeast chromosomal DNA for PFGE and general PFGE procedures and will introduce a PFGE method to monitor the DNA replication fork progression and DNA double-strand breaks (DSBs).

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Spp1 is the H3K4me3 reader subunit of the Set1 complex (COMPASS/Set1C) that contributes to the mechanism by which meiotic DNA break sites are mechanistically selected. We previously proposed a model in which Spp1 interacts with H3K4me3 and the chromosome axis protein Mer2 that leads to DSB formation. Here we show that spatial interactions of Spp1 and Mer2 occur independently of Set1C. Spp1 exhibits dynamic chromatin binding features during meiosis, with many de novo appearing and disappearing binding sites. Spp1 chromatin binding dynamics depends on its PHD finger and Mer2-interacting domain and on modifiable histone residues (H3R2/K4). Remarkably, association of Spp1 with Mer2 axial sites reduces the effective turnover rate and diffusion coefficient of Spp1 upon chromatin binding, compared with other Set1C subunits. Our results indicate that "chromosomal turnover rate" is a major molecular determinant of Spp1 function in the framework of meiotic chromatin structure that prepares recombination initiation sites for break formation., (© 2018 Karányi et al.)

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11-Rad50-Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre11 2 -Rad50 2 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid., Competing Interests: The authors declare no conflict of interest.

Meiotic recombination is initiated by programmed formation of DNA double strand breaks (DSBs), which are mainly formed at recombination hotspots. Meiotic DSBs require multiple proteins including the conserved protein Spo11 and its cofactors, and are influenced by chromatin structure. For example, local chromatin around hotspots directly impacts DSB formation. Moreover, DSB is proposed to occur in a higher-order chromatin architecture termed 'axis-loop', in which many loops protrude from cohesin-enriched axis. However, still much remains unknown about how meiotic DSBs are generated in chromatin. Here, we show that the conserved histone H2A variant H2A.Z promotes meiotic DSB formation in fission yeast. Detailed investigation revealed that H2A.Z is neither enriched around hotspots nor axis sites, and that transcript levels of DSB-promoting factors were maintained without H2A.Z. Moreover, H2A.Z appeared to be dispensable for chromatin binding of meiotic cohesin. Instead, in H2A.Z-lacking mutants, multiple proteins involved in DSB formation, such as the fission yeast Spo11 homolog and its regulators, were less associated with chromatin. Remarkably, nuclei were more compact in the absence of H2A.Z. Based on these, we propose that fission yeast H2A.Z promotes meiotic DSB formation partly through modulating chromosome architecture to enhance interaction between DSB-related proteins and cohesin-loaded chromatin., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of adenosine triphosphate hydrolysis-dependent translocation along double-stranded DNA. Condensin's translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

DNA replication of circular genomes generates physically interlinked or catenated sister DNAs. These are resolved through transient DNA fracture by type II topoisomerases to permit chromosome segregation during cell division. Topoisomerase II is similarly required for linear chromosome segregation, suggesting that linear chromosomes also remain intertwined following DNA replication. Indeed, chromosome resolution defects are a frequent cause of chromosome segregation failure and consequent aneuploidies. When and where intertwines arise and persist along linear chromosomes are not known, owing to the difficulty of demonstrating intertwining of linear DNAs. Here, we used excision of chromosomal regions as circular "loop outs" to convert sister chromatid intertwines into catenated circles. This revealed intertwining at replication termination and cohesin-binding sites, where intertwines are thought to arise and persist but not to a greater extent than elsewhere in the genome. Intertwining appears to spread evenly along chromosomes but is excluded from heterochromatin. We found that intertwines arise before replication termination, suggesting that replication forks rotate during replication elongation to dissipate torsion ahead of the forks. Our approach provides previously inaccessible insight into the topology of eukaryotic chromosomes and illuminates a process critical for successful chromosome segregation., (© 2017 Mariezcurrena and Uhlmann; Published by Cold Spring Harbor Laboratory Press.)

The question of how self-interacting chromatin domains in interphase chromosomes are structured and generated dominates current discussions on eukaryotic chromosomes. Numerical simulations using standard polymer models have been helpful in testing the validity of various models of chromosome organization. Experimental contact maps can be compared with simulated contact maps and thus verify how good is the model. With increasing resolution of experimental contact maps, it became apparent though that active processes need to be introduced into models to recapitulate the experimental data. Since transcribing RNA polymerases are very strong molecular motors that induce axial rotation of transcribed DNA, we present here models that include such rotational motors. We also include into our models swivels and sites for intersegmental passages that account for action of DNA topoisomerases releasing torsional stress. Using these elements in our models, we show that transcription-induced supercoiling generated in the regions with divergent-transcription and supercoiling relaxation occurring between these regions are sufficient to explain formation of self-interacting chromatin domains in chromosomes of fission yeast (S. pombe)., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Structural maintenance of chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids, while condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate that this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead, it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes.

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4) 2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

Sister chromatid intertwines (SCIs), or catenanes, are topological links between replicated chromatids that interfere with chromosome segregation. The formation of SCIs is thought to be a consequence of fork swiveling during DNA replication, and their removal is thought to occur because of the intrinsic feature of type II topoisomerases (Top2) to simplify DNA topology. Here, we report that SCIs are also formed independently of DNA replication during G 2 /M by Top2-dependent concatenation of cohesed chromatids due to their physical proximity. We demonstrate that, in contrast to G 2 /M, Top2 removes SCIs from cohesed chromatids at the anaphase onset. Importantly, SCI removal in anaphase requires condensin and coincides with the hyperactivation of condensin DNA supercoiling activity. This is consistent with the longstanding proposal that condensin provides a bias in Top2 function toward decatenation. A comprehensive model for the formation and resolution of toxic SCI entanglements on eukaryotic genomes is proposed., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The number and distribution of meiosis crossover (CO) events on each bivalent are strictly controlled by multiple mechanisms to assure proper chromosome segregation during the first meiotic division. In Saccharomyces cerevisiae, Slx4 is a multi-functional scaffold protein for structure-selective endonucleases, such as Slx1 and Rad1 (which are involved in DNA damage repair), and is also a negative regulator of the Rad9-dependent signaling pathway with Rtt107 Slx4 has been believed to play only a minor role in meiotic recombination. Here, we report that Slx4 is involved in proper intrachromosomal distribution of meiotic CO formation, especially in regions near centromeres. We observed an increase in uncontrolled CO formation only in a region near the centromere in the slx4∆ mutant. Interestingly, this phenomenon was not observed in the slx1∆, rad1∆, or rtt107∆ mutants. In addition, we observed a reduced number of DNA double-strand breaks (DSBs) and altered meiotic DSB distribution on chromosomes in the slx4∆ mutant. This suggests that the multi-functional Slx4 is required for proper CO formation and meiotic DSB formation., (Copyright © 2016 Higashide and Shinohara.)

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Condensin is not only responsible for chromosome condensation, but is also involved in double-strand break (DSB) processing in the cell cycle. During meiosis, the condensin complex serves as a component of the meiotic chromosome axis, and mediates both proper assembly of the synaptonemal complex and DSB repair, in order to ensure proper homologous chromosome segregation. Here, we used the budding yeast Saccharomyces cerevisiae to show that condensin participates in a variety of chromosome organization processes and exhibits crucial molecular functions that contribute to meiotic recombination during meiotic prophase I. We demonstrate that Ycs4 is required for efficient DSB formation and establishing homolog bias at the early stage of meiotic prophase I, which allows efficient formation of interhomolog recombination products. In the Ycs4 meiosis-specific allele (ycs4S), interhomolog products were formed at substantial levels, but with the same reduction in crossovers and noncrossovers. We further show that, in prophase chromosomal events, ycs4S relieved the defects in the progression of recombination interactions induced as a result of the absence of Rec8. These results suggest that condensin is a crucial coordinator of the recombination process and chromosome organization during meiosis.

The nucleolus is considered to be a stress sensor and rDNA-based regulation of cellular senescence and longevity has been proposed. However, the role of rDNA in the maintenance of genome integrity has not been investigated in detail. Using genomically diverse industrial yeasts as a model and array-based comparative genomic hybridization (aCGH), we show that chromosome level may be balanced during passages and as a response to alcohol stress that may be associated with changes in rDNA pools. Generation- and ethanol-mediated changes in genes responsible for protein and DNA/RNA metabolism were revealed using next-generation sequencing. Links between redox homeostasis, DNA stability, and telomere and nucleolus states were also established. These results suggest that yeast genome is dynamic and chromosome homeostasis may be controlled by rDNA.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation., (© 2014 Samel et al.; Published by Cold Spring Harbor Laboratory Press.)

Eukaryotic topoisomerases I (topo I) and II (topo II) relax the positive (+) and negative (-) DNA torsional stress (TS) generated ahead and behind the transcription machinery. It is unknown how this DNA relaxation activity is regulated and whether (+) and (-)TS are reduced at similar rates. Here, we used yeast circular minichromosomes to conduct the first comparative analysis of topo I and topo II activities in relaxing chromatin under (+) and (-)TS. We observed that, while topo I relaxed (+) and (-)TS with similar efficiency, topo II was more proficient and relaxed (+)TS more quickly than (-)TS. Accordingly, we found that the relaxation rate of (+)TS by endogenous topoisomerases largely surpassed that of (-)TS. We propose a model of how distinct conformations of chromatin under (+) and (-)TS may produce this unbalanced relaxation of DNA. We postulate that, while quick relaxation of (+)TS may facilitate the progression of RNA and DNA polymerases, slow relaxation of (-)TS may serve to favor DNA unwinding and other structural transitions at specific regions often required for genomic transactions., (© 2014 The Authors.)

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

The establishment and maintenance of higher-order structure at centromeres is essential for accurate chromosome segregation. The monopolin complex is thought to cross-link multiple kinetochore complexes to prevent merotelic attachments that result in chromosome missegregation. This model is based on structural analysis and the requirement that monopolin execute mitotic and meiotic chromosome segregation in Schizosaccharomyces pombe, which has more than one kinetochore-microtubule attachment/centromere, and co-orient sister chromatids in meiosis I in Saccharomyces cerevisiae. Recent data from S. pombe suggest an alternative possibility: that the recruitment of condensin is the primary function of monopolin. Here we test these models using the yeast Candida albicans. C. albicans cells lacking monopolin exhibit defects in chromosome segregation, increased distance between centromeres, and decreased stability of several types of repeat DNA. Of note, changing kinetochore-microtubule copy number from one to more than one kinetochore-microtubule/centromere does not alter the requirement for monopolin. Furthermore, monopolin recruits condensin to C. albicans centromeres, and overexpression of condensin suppresses chromosome segregation defects in strains lacking monopolin. We propose that the key function of monopolin is to recruit condensin in order to promote the assembly of higher-order structure at centromere and repetitive DNA.

The structure of the nucleosome has been solved at atomic resolution, and the genome-wide nucleosome positions have been clarified for the budding yeast Saccharomyces cerevisiae. However, the genome-wide three-dimensional arrangement of nucleosomal arrays in the nucleus remains unclear. Several studies simulated overall interphase chromosome architectures by introducing the putative persistence length of the controversial 30-nm chromatin fibres into the modelling and using data-fitting approaches. However, the genome-folding mechanism still could not be linked with the chromosome shapes, to identify which structures or properties of chromatin fibres or DNA sequences determine the overall interphase chromosome architectures. Here we demonstrate that the paths of nucleosomal arrays and the chromatin architectures themselves are determined principally by the physical properties of genomic DNA and the nucleus size in yeast. We clarified the flexibilities and persistence lengths of all linker DNAs of the organism, deduced their spatial expanses and simulated the architectures of all 16 interphase chromosomes in the nucleus, at a resolution of beads-on-a-string chromatin fibre. For the average spatial distance between two given loci in a chromosome, the model predictions agreed well with all experimental data reported to date. These findings suggest a general mechanism underlying the folding of eukaryotic genomes into interphase chromosomes.

Telomere repeat-like sequences at DNA double-strand breaks (DSBs) inhibit DNA damage signaling and serve as seeds to convert DSBs to new telomeres in mutagenic chromosome healing pathways. We find here that the response to seed-containing DSBs differs fundamentally between budding yeast (Saccharomyces cerevisiae) cells that maintain their telomeres via telomerase and so-called postsenescence survivors that use recombination-based alternative lengthening of telomere (ALT) mechanisms. Whereas telomere seeds are efficiently elongated by telomerase, they remain remarkably stable without de novo telomerization or extensive end resection in telomerase-deficient (est2Δ, tlc1Δ) postsenescence survivors. This telomere seed hyper-stability in ALT cells is associated with, but not caused by, prolonged DNA damage checkpoint activity (RAD9, RAD53) compared to telomerase-positive cells or presenescent telomerase-negative cells. The results indicate that both chromosome healing and anticheckpoint activity of telomere seeds are suppressed in yeast models of ALT pathways.

Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

DNA double-strand break (DSB) repair occurring in repeated DNA sequences often leads to the generation of chromosomal rearrangements. Homologous recombination normally ensures a faithful repair of DSBs through a mechanism that transfers the genetic information of an intact donor template to the broken molecule. When only one DSB end shares homology to the donor template, conventional gene conversion fails to occur and repair can be channeled to a recombination-dependent replication pathway termed break-induced replication (BIR), which is prone to produce chromosome non-reciprocal translocations (NRTs), a classical feature of numerous human cancers. Using a newly designed substrate for the analysis of DSB-induced chromosomal translocations, we show that Mus81 and Yen1 structure-selective endonucleases (SSEs) promote BIR, thus causing NRTs. We propose that Mus81 and Yen1 are recruited at the strand invasion intermediate to allow the establishment of a replication fork, which is required to complete BIR. Replication template switching during BIR, a feature of this pathway, engenders complex chromosomal rearrangements when using repeated DNA sequences dispersed over the genome. We demonstrate here that Mus81 and Yen1, together with Slx4, also promote template switching during BIR. Altogether, our study provides evidence for a role of SSEs at multiple steps during BIR, thus participating in the destabilization of the genome by generating complex chromosomal rearrangements., Competing Interests: The authors have declared that no competing interests exist.

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv.

Site-specific recombinases have been harnessed for a variety of genetic manipulations involving the gain, loss, or rearrangement of genomic DNA in a variety of organisms. The enzymes have been further exploited in the model eukaryote Saccharomyces cerevisiae for mechanistic studies involving chromosomal context. In these cases, a chromosomal element of interest is converted into a DNA circle within living cells, thereby uncoupling the element from neighboring regulatory sequences, obligatory chromosomal events, and other context-dependent effects that could alter or mask intrinsic functions of the element. In this chapter, I discuss general considerations in using site-specific recombination to create DNA circles in yeast and the specific application of the R recombinase.

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.

Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.

We present a mathematical model for the spatial dynamics of DNA replication. Using this model we determine the probability distribution for the time at which each chromosomal position is replicated. From this we show, contrary to previous reports, that mean replication time curves cannot be used to directly determine origin parameters. We demonstrate that the stochastic nature of replication dynamics leaves a clear signature in experimentally measured population average data, and we show that the width of the activation time probability distribution can be inferred from this data. Our results compare favorably with experimental measurements in Saccharomyces cerevisae.

Cohesin is a member of the Smc family of protein complexes that mediates higher-order chromosome structure by tethering different regions of chromatin. We present a new in vitro system that assembles cohesin-DNA complexes with in vivo properties. The assembly of these physiological salt-resistant complexes requires the cohesin holo-complex, its ability to bind ATP, the cohesin loader Scc2p and a closed DNA topology. Both the number of cohesin molecules bound to the DNA substrate and their distribution on the DNA substrate are limited. Cohesin and Scc2p bind preferentially to cohesin associated regions (CARs), DNA sequences with enriched cohesin binding in vivo. A subsequence of CARC1 promotes cohesin binding to neighboring sequences within CARC1. The enhancer-like function of this sequence is validated by in vivo deletion analysis. By demonstrating the physiological relevance of these in vitro assembled cohesin-DNA complexes, we establish our in vitro system as a powerful tool to elucidate the mechanism of cohesin and other Smc complexes.

The multisubunit condensin complex is essential for the structural organization of eukaryotic chromosomes during their segregation by the mitotic spindle, but the mechanistic basis for its function is not understood. To address how condensin binds to and structures chromosomes, we have isolated from Saccharomyces cerevisiae cells circular minichromosomes linked to condensin. We find that either linearization of minichromosome DNA or proteolytic opening of the ring-like structure formed through the connection of the two ATPase heads of condensin's structural maintenance of chromosomes (SMC) heterodimer by its kleisin subunit eliminates their association. This suggests that condensin rings encircle chromosomal DNA. We further show that release of condensin from chromosomes by ring opening in dividing cells compromises the partitioning of chromosome regions distal to centromeres. Condensin hence forms topological links within chromatid arms that provide the arms with the structural rigidity necessary for their segregation.

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.

The assembly of a protective cap onto the telomeres of eukaryotic chromosomes suppresses genomic instability through inhibition of DNA repair activities that normally process accidental DNA breaks. We show here that the essential Cdc13-Stn1-Ten1 complex is entirely dispensable for telomere protection in non-dividing cells. However, Yku and Rap1 become crucially important for this function in these cells. After inactivation of Yku70 in G1-arrested cells, moderate but significant telomere degradation occurs. As the activity of cyclin-dependent kinases (CDK) promotes degradation, these results suggest that Yku stabilizes G1 telomeres by blocking the access of CDK1-independent nucleases to telomeres. The results indeed show that both Exo1 and the Mre11/Rad50/Xrs2 complex are required for telomeric resection after Yku loss in non-dividing cells. Unexpectedly, both asynchronously growing and quiescent G0 cells lacking Rap1 display readily detectable telomere degradation, suggesting an earlier unanticipated function for this protein in suppression of nuclease activities at telomeres. Together, our results show a high flexibility of the telomeric cap and suggest that distinct configurations may provide for efficient capping in dividing versus non-dividing cells.

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation., (Copyright 2009 Elsevier B.V. All rights reserved.)

How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin.

Positioned nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we have generated the first genome-wide nucleosome positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome wide. Using this resource, we found surprising differences from the previously published nucleosome organization of the distantly related yeast Saccharomyces cerevisiae. DNA sequence guides nucleosome positioning differently: for example, poly(dA-dT) elements are not enriched in S. pombe nucleosome-depleted regions. Regular nucleosomal arrays emanate more asymmetrically-mainly codirectionally with transcription-from promoter nucleosome-depleted regions, but promoters harboring the histone variant H2A.Z also show regular arrays upstream of these regions. Regular nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs and requires a remodeler, Mit1, that is conserved in humans but is not found in S. cerevisiae. Nucleosome positioning mechanisms are evidently not universal but evolutionarily plastic.

The association of chromosomes with each other and other nuclear components plays a critical role in nuclear organization and Genome function. Here, using a novel and generally applicable methodology (Genome conformation capture [GCC]), we reveal the network of chromosome interactions for the yeast Saccharomyces cerevisiae. Inter- and intra-chromosomal interactions are non-random and the number of interactions per open reading frame depends upon the dispensability of the gene product. Chromosomal interfaces are organized and provide evidence of folding within chromosomes. Interestingly, the genomic connections also involve the 2 microm plasmid and the mitochondrial genome. Mitochondrial interaction partners include genes of alpha-proteobacterial origin and the ribosomal DNA. Organization of the 2 microm plasmid aligns two inverted repeats (IR1 and IR2) and displays the stability locus on a prominent loop thus making it available for plasmid clustering. Our results form the first global map of chromosomal interactions in a eukaryotic nucleus and demonstrate the highly connected nature of the yeast genome. These results have significant implications for understanding eukaryotic genome organization.

Accurate chromosome segregation depends on the kinetochore, which is the complex of proteins that link microtubules to centromeric DNA. The kinetochore of the budding yeast Saccharomyces cerevisiae consists of more than 80 proteins assembled on a 125-bp region of DNA. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1-DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, bi-orient sister kinetochores at metaphase during the mitotic cycle, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers assembly of the complex structure that directs mitotic chromosome segregation.

DNA replication in eukaryotes is considered to proceed according to a precise program in which each chromosomal region is duplicated in a defined temporal order. However, recent studies reveal an intrinsic temporal disorder in the replication of yeast chromosome VI. Here we provide a model of the chromosomal duplication to study the temporal sequence of origin activation in budding yeast. The model comprises four parameters that influence the DNA replication system: the lengths of the chromosomes, the explicit chromosomal positions for all replication origins as well as their distinct initiation times and the replication fork migration rate. The designed model is able to reproduce the available experimental data in form of replication profiles. The dynamics of DNA replication was monitored during simulations of wild type and randomly perturbed replication conditions. Severe loss of origin function showed only little influence on the replication dynamics, so systematic deletions of origins (or loss of efficiency) were simulated to provide predictions to be tested experimentally. The simulations provide new insights into the complex system of DNA replication, showing that the system is robust to perturbation, and giving hints about the influence of a possible disordered firing.

DNA double-strand breaks (DSBs) are acutely hazardous for cells, as they can cause genome instability. DSB repair involves the sequential recruitment of repair factors to the DSBs, followed by Rad51-mediated homology probing, DNA synthesis, and ligation. However, little is known about how cells react if no homology is found and DSBs persist. Here, by monitoring a single persistent DNA break, we show that, following DNA resection and RPA recruitment, Rad51 spreads chromosome-wide bidirectionally from the DSB but selectively only on the broken chromosome. Remarkably, the persistent DSB is later fixed to the nuclear periphery in a process that requires Rad51, the histone variant H2A.Z, its SUMO modification, and the DNA-damage checkpoint. Indeed, H2A.Z is deposited close to the break early but transiently and directs DNA resection, single DSB-induced checkpoint activation, and DSB anchoring. Thus, a persistent DSB induces a multifaceted response, which is linked to a specific chromatin mark.

The density transfer technique is a valuable tool to examine the progression of individual DNA replication forks. It is based on the transfer of cells from a medium containing dense isotopes to a medium with light (normal) isotopes (or vice versa), to obtain DNA sequences hybrid in density that can be identified as replicated molecules. Using specific DNA probes along a chromosome, the dense isotope transfer method allows determining the extent of replication at any position of a replicon and the rate of replication fork progression. In the eukaryotic model budding yeast, this technique has been useful to establish a role for different proteins during the elongation of chromosomal replication and to analyze the movement and stability of DNA replication forks under different experimental conditions.

Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observed, and the hypothesis to be tested. In this chapter, we review three broad types of visualization: whole cell fluorescence or immunofluorescence, which is useful for questions of timing and chromatin association; nuclear spreads, which provide greater resolution within the chromatin for colocalization and region-specific effects; and chromatin fibers, which allow observation of labeled proteins and newly synthesized DNA on a linear chromosome. We discuss applications of these protocols and some considerations for choosing methods and fluorophores.