The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. 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.

Sequence-dependent nucleosome formation in trinucleotide repeats evaluated by in vivo chemical mapping.

Trinucleotide repeat sequences (TRSs), consisting of 10 unique classes of repeats in DNA, are members of microsatellites and abundantly and non-randomly distributed in many eukaryotic genomes. The lengths of TRSs are mutable, and the expansions of several TRSs are implicated in hereditary neurological diseases. However, the underlying causes of the biased distribution and the dynamic properties of TRSs in the genome remain elusive. Here, we examined the effects of TRSs on nucleosome formation in vivo by histone H4-S47C site-directed chemical cleavages, using well-defined yeast minichromosomes in which each of the ten TRS classes resided in the central region of a positioned nucleosome. We showed that (AAT)12 and (ACT)12 act as strong nucleosome-promoting sequences, while (AGG)12 and (CCG)12 act as nucleosome-excluding sequences in vivo. The local histone binding affinity scores support the idea that nucleosome formation in TRSs, except for (AGG)12, is mainly determined by the affinity for the histone octamers. Overall, our study presents a framework for understanding the nucleosome-forming abilities of TRSs.

Genetic and Molecular Approaches to Study Chromosomal Breakage at Secondary Structure-Forming Repeats.

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs. Then, we detail an approach to monitor chromosomal DSBs by Southern blot hybridization. Finally, we describe how to define the molecular structure of DSBs. We provide, as an example, the analysis of chromosomal fragility at a reporter system containing unstable Alu-inverted repeats. By using these approaches, any DNA sequence motif can be assessed for its breakage potential and ability to drive genome instability.

Spatial inter-centromeric interactions facilitated the emergence of evolutionary new centromeres.

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

In vivo chromatin organization on native yeast telomeric regions is independent of a cis-telomere loopback conformation.

BACKGROUND: DNA packaging into chromatin regulates all DNA-related processes and at chromosomal ends could affect both essential functions of telomeres: protection against DNA damage response and telomere replication. Despite this primordial role of chromatin, little is known about chromatin organization, and in particular about nucleosome positioning on unmodified subtelomere-telomere junctions in Saccharomyces cerevisiae. RESULTS: By ChEC experiments and indirect end-labeling, we characterized nucleosome positioning as well as specialized protein-DNA associations on most subtelomere-telomere junctions present in budding yeast. The results show that there is a relatively large nucleosome-free region at chromosome ends. Despite the absence of sequence homologies between the two major classes of subtelomere-telomere junctions (i.e.: Y'-telomeres and X-telomeres), all analyzed subtelomere-telomere junctions show a terminal nucleosome-free region just distally from the known Rap1-covered telomeric repeats. Moreover, previous evidence suggested a telomeric chromatin fold-back structure onto subtelomeric areas that supposedly was implicated in chromosome end protection. The in vivo ChEC method used herein in conjunction with several proteins in a natural context revealed no evidence for such structures in bulk chromatin. CONCLUSIONS: Our study allows a structural definition of the chromatin found at chromosome ends in budding yeast. This definition, derived with direct in vivo approaches, includes a terminal area that is free of nucleosomes, certain positioned nucleosomes and conserved DNA-bound protein complexes. This organization of subtelomeric and telomeric areas however does not include a telomeric cis-loopback conformation. We propose that the observations on such fold-back structures may report rare and/or transient associations and not stable or constitutive structures.

Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners.

Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.

Monitoring of DNA Replication and DNA Double-Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis (PFGE).

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).

Pulsed-Field Gel Electrophoresis for Detecting Chromosomal DNA Breakage in Fission Yeast.

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.

Principles of meiotic chromosome assembly revealed in S. cerevisiae.

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we use Saccharomyces cerevisiae to explore how this elaborate three-dimensional chromosome organisation is linked to genomic sequence. As cells enter meiosis, we observe that strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion with growth limited by barriers, in which a heterogeneous population of expanding loops develop along the chromosome. Importantly, CTCF, the factor that imposes similar features in mammalian interphase, is absent in S. cerevisiae, suggesting alternative mechanisms of barrier formation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

Nucleosome positions alone can be used to predict domains in yeast chromosomes.

We use molecular dynamics simulations based on publicly available micrococcal nuclease sequencing data for nucleosome positions to predict the 3D structure of chromatin in the yeast genome. Our main aim is to shed light on the mechanism underlying the formation of chromosomal interaction domains, chromosome regions of around 0.5 to 10 kbp which show enriched self-interactions, which were experimentally observed in recent MicroC experiments (importantly these are at a different length scale from the 100- to 1,000-kbp-sized domains observed in higher eukaryotes). We show that the sole input of nucleosome positioning data is already sufficient to determine the patterns of chromatin interactions and domain boundaries seen experimentally to a high degree of accuracy. Since the nucleosome spacing so strongly affects the larger-scale domain structure, we next examine the genome-wide linker-length distribution in more detail, finding that it is highly irregular and varies in different genomic regions such as gene bodies, promoters, and active and inactive genes. Finally we use our simple simulation model to characterize in more detail how irregular nucleosome spacing may affect local chromatin structure.

Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.

The Rabl configuration limits topological entanglement of chromosomes in budding yeast.

The three dimensional organization of genomes remains mostly unknown due to their high degree of condensation. Biophysical studies predict that condensation promotes the topological entanglement of chromatin fibers and the inhibition of function. How organisms balance between functionally active genomes and a high degree of condensation remains to be determined. Here we hypothesize that the Rabl configuration, characterized by the attachment of centromeres and telomeres to the nuclear envelope, helps to reduce the topological entanglement of chromosomes. To test this hypothesis we developed a novel method to quantify chromosome entanglement complexity in 3D reconstructions obtained from Chromosome Conformation Capture (CCC) data. Applying this method to published data of the yeast genome, we show that computational models implementing the attachment of telomeres or centromeres alone are not sufficient to obtain the reduced entanglement complexity observed in 3D reconstructions. It is only when the centromeres and telomeres are attached to the nuclear envelope (i.e. the Rabl configuration) that the complexity of entanglement of the genome is comparable to that of the 3D reconstructions. We therefore suggest that the Rabl configuration is an essential player in the simplification of the entanglement of chromatin fibers.

Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome.

Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet it is well established that host strain background is just as important for determining productivity. Here we report that in vivo combinatorial genomic rearrangement of Saccharomyces cerevisiae yeast with a synthetic chromosome V can rapidly generate new, improved host strains with genetic backgrounds favourable to diverse heterologous pathways, including those for violacein and penicillin biosynthesis and for xylose utilisation. We show how the modular rearrangement of synthetic chromosomes by SCRaMbLE can be easily determined using long-read nanopore sequencing and we explore experimental conditions that optimise diversification and screening. This synthetic genome approach to metabolic engineering provides productivity improvements in a fast, simple and accessible way, making it a valuable addition to existing strain improvement techniques.

Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods.

Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed "SCRaMbLE-in") to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of ß-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

Heterozygous diploid and interspecies SCRaMbLEing.

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II.

Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.