Convergent genes shape budding yeast pericentromeres.

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.

Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage.

BACKGROUND: The main function of telomerase is at the telomeres but under adverse conditions telomerase can bind to internal regions causing deleterious effects as observed in cancer cells. RESULTS: By mapping the global occupancy of the catalytic subunit of telomerase (Est2) in the budding yeast Saccharomyces cerevisiae, we reveal that it binds to multiple guanine-rich genomic loci, which we termed "non-telomeric binding sites" (NTBS). We characterize Est2 binding to NTBS. Contrary to telomeres, Est2 binds to NTBS in G1 and G2 phase independently of Est1 and Est3. The absence of Est1 and Est3 renders telomerase inactive at NTBS. However, upon global DNA damage, Est1 and Est3 join Est2 at NTBS and telomere addition can be observed indicating that Est2 occupancy marks NTBS regions as particular risks for genome stability. CONCLUSIONS: Our results provide a novel model of telomerase regulation in the cell cycle using internal regions as "parking spots" of Est2 but marking them as hotspots for telomere addition.

Are SMC Complexes Loop Extruding Factors? Linking Theory With Fact.

The extreme length of chromosomal DNA requires organizing mechanisms to both promote functional genetic interactions and ensure faithful chromosome segregation when cells divide. Microscopy and genome-wide contact frequency analyses indicate that intra-chromosomal looping of DNA is a primary pathway of chromosomal organization during all stages of the cell cycle. DNA loop extrusion has emerged as a unifying model for how chromosome loops are formed in cis in different genomic contexts and cell cycle stages. The highly conserved family of SMC complexes have been found to be required for DNA cis-looping and have been suggested to be the enzymatic core of loop extruding machines. Here, the current body of evidence available for the in vivo and in vitro action of SMC complexes is discussed and compared to the predictions made by the loop extrusion model. How SMC complexes may differentially act on chromatin to generate DNA loops and how they could work to generate the dynamic and functionally appropriate organization of DNA in cells is explored.

Recombination-restarted replication makes inverted chromosome fusions at inverted repeats.

Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

A winged helix domain in human MUS81 binds DNA and modulates the endonuclease activity of MUS81 complexes.

The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.

Hi-C2B: Optimized Detection of Chromosomal Contacts Within Synchronized Meiotic S. cerevisiae Cells.

Hi-C, a genome-wide chromosome conformation capture assay, is a powerful tool used to study three-dimensional genome organization by converting physical pairwise interactions into counts of pairwise interactions. To study the many temporally regulated facets of meiotic recombination in S. cerevisiae, the Hi-C assay must be robust such that fine- and wide-scale comparisons between genetic datasets can be made. Here we describe an updated protocol for Hi-C (Hi-C2B) that generates reproducible libraries of interaction data with low noise and for a relatively low cost.

Preparation of Cell Cycle-Synchronized Saccharomyces cerevisiae Cells for Hi-C.

The chromosome organization activities of SMC complexes are crucial for correct gene expression and genetic inheritance in cells. Hi-C assays have revealed previously unsuspected levels of chromosome structure, with different types of chromosome structure facilitating function at different stages of the cell cycle. Elucidating how SMC complexes regulate these distinct types of organization is currently a key question in molecular biology.The range of genetic tools and the small genome size of the budding yeast Saccharomyces cerevisiae make it an ideal tool for studying how SMC complexes control chromosome structure in eukaryotic cells. A crucial advantage of S. cerevisiae over other systems is that large populations of cells can be easily arrested at distinct stages of the cell cycle and SMC gene function specifically ablated in the synchronized cells. Here we describe methods to prepare synchronously cell cycle-arrested populations of genetically modified S. cerevisiae cells for Hi-C analysis.

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.

SMC complexes differentially compact mitotic chromosomes according to genomic context.

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.