Entangling and disentangling mitotic chromosomes.

The DNA topological challenges generated by cellular manipulation of extremely long DNA fibers remain poorly understood. In this issue of Molecular Cell, Hildebrand et al.1 describe how mitotic chromosomes are self entangled and that disentanglement requires TOP2 activity in late mitosis.

Cohesin Causes Replicative DNA Damage by Trapping DNA Topological Stress.

DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae, we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological-stress-linked damage in these regions. At cohesin-enriched centromeres, cohesin activity causes the accumulation of DNA damage, RF rotation, and pre-catenation, confirming that cohesin-dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA, cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that, as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.

Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.

The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

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.

Cryo-EM structure of the Smc5/6 holo-complex.

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress.

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

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.

A systematic review of pre-hospital shoulder reduction techniques for anterior shoulder dislocation and the effect on patient return to function.

PURPOSE: The majority of acute anterior shoulder dislocations are sustained during sports and wilderness activities. The management of acute dislocations in the pre-hospital setting is currently without guidelines based on the evidence. The study aims to assess the risk of acute complications in pre-hospital shoulder reduction and identify which pre-hospital reduction technique has the highest success rate in the published literature. METHODS: The involved databases were Allied and Complementary Medicine, CENTRAL, CINAHL, Cochrane Database of Systematic Reviews, Embase, Europe PMC, Ovid MEDLINE®, Pedro, Proquest, Trip, and World Health Organization International Clinical Trials Registry platform. Only original research of high methodological quality was included, which was defined by the recently developed assessment tool-assessing the methodological quality of published papers (AMQPP) and investigated the management of acute anterior shoulder dislocations in the pre-hospital setting. RESULTS: Two hundred and ninety-eight articles were identified and screened. A full text review was performed on 40 articles. Four articles published between 2015 and 2018 met the inclusion criteria. A total of 181 patients were included with the study duration ranging from 6 to 60 months. All studies reported zero immediate complication following pre-hospital reduction and there were no documented subsequent adverse events regardless of the technique used. Prompt resolution of neurological symptoms was observed following the early and successful pre-hospital reduction. First attempt success rate, when performed by skilled practitioners, ranged from 72.3% to 94.9%. CONCLUSION: Pre-hospital shoulder reduction appears to be a safe and feasible option when carried out with the appropriate expertise. A novel reduction technique adapted from the mountain medicine diploma course at the University of Paris North was found to have the highest first attempt reduction success rate of 94.9%. Other techniques described in the literature included Hippocratic, Stimson's, Counter-traction and external rotation with the success rates ranging from 54% to 71.7%.

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.

Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast.

Type II topoisomerases are essential for resolving topologically entwined double-stranded DNA. Although anti-topoisomerase 2 (Top2) drugs are clinically important antibiotics and chemotherapies, to our knowledge, the mechanisms of cell killing by Top2 depletion and inactivation have never been directly compared. We show that depletion of Top2 protein from budding yeast cells prevents DNA decatenation during S phase. Cells complete DNA replication and enter the ensuing mitosis on schedule, suffering extensive chromosome missegregation. Cytokinesis through incompletely segregated chromosomes causes lethal DNA damage. By contrast, expression of catalytically inactive Top2 causes a stable G2 arrest requiring an intact DNA damage checkpoint. Checkpoint activation correlates with an inability to complete DNA replication, resulting in hypercatenated, gapped daughter DNA molecules. Thus, Top2 depletion and inactivation kill cells by different mechanisms, which has implications for understanding the nature of the catenation checkpoint, how DNA replication terminates, how anti-Top2 drugs work, and how new drugs might be designed.

A model for chromosome condensation based on the interplay between condensin and topoisomerase II.

The compaction of chromatin that occurs when cells enter mitosis is probably the most iconic process of dividing cells. Mitotic chromosomal compaction or 'condensation' is functionally linked to resolution of chromosomal intertwines, transcriptional shut-off and complete segregation of chromosomes. At present, understanding of the molecular events required to convert interphase chromatin into mitotic chromosomes is limited. Here, we review recent advances in the field, focusing on potential chromosomal compaction mechanisms and their importance to chromosome segregation. We propose a model of how metaphase chromosomes could be shaped based on the enzymatic activities of condensin and topoisomerase II in overwinding and relaxation of the DNA fiber during mitosis. We suggest that condensin overwinding is an important requirement for intertwine resolution by topoisomerase II and, together with the inhibition of transcription, contributes to cytological mitotic chromosome appearance or 'condensation'.

Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

The Validation of Surgical Simulators: A Technical Report on Current Validation Terminology as a Reference for Future Research.

In recent years, surgical trainees have been exposed to a lower volume of operative procedures. In part, this is due to the reduction in working hours and further disruption by the coronavirus disease 2019 pandemic. Much has been done to develop the techniques of surgical skill training outside of the operating theatre. Simulation-based interventions must undergo a process of validation to assess their appropriateness and effectiveness for use in training. The terminology of validation within current literature, however, has not evolved in line with the education community, resulting in varying definitions for the same phrase across domains. This can result in confusion and misinterpretation among researchers and surgeons working within this domain. This technical report describes the "types of validity" definitions used in the traditional framework of surgical simulation literature and the contemporary, unitary framework of validity adopted by educationalist theorists. There is a clear overlap between the traditional "types of validity" and the contemporary, unitary framework. The divergence in the use of those definitions seems, at least partly, influenced by the context of the investigations being conducted. By utilising the contemporary definitions, authors may have struggled to provide the evidence required to justify the use of the multitude of surgical skill simulators developed in the recent past. This report has provided an overview of the current terminology within the validation frameworks and can be used as a reference for future surgical simulation research.

Nuclear organisation and gene expression.

The development of increasingly sophisticated tools to track chromosomes and proteins in living cells offers the possibility of visualising gene regulation in the nucleus with minimal distortion. This, in conjunction with powerful genetic approaches available in yeast, is beginning to allow functional definition of nuclear "compartments".

The FAST Workstation Shows Construct Validity and Participant Endorsement.

PURPOSE: To determine in what way the proposed simulation-based intervention (SBI) is an effective intervention for use in basic arthroscopic skills training. METHODS: Twenty candidates were recruited and grouped according to experience. Performance metrics included the time to activity completion, errors made, and Global Rating Scale score. Qualitative data were collected using a structured questionnaire. RESULTS: Performance on the SBI differed depending on previous arthroscopic training received. Performance on the simulator differed between groups to a statistically significant level regarding time to completion. A difference was also present between participants with no previous training and those with previous training when assessed using the Global Rating Scale. The SBI was deemed acceptable, user-friendly, and realistic. Participants practicing at the expert level believe that such an SBI would be beneficial in developing basic arthroscopic skills. CONCLUSIONS: The results of this study provide evidence that the use of an SBI consisting of a benchtop workstation, laptop viewing platform, 30° arthroscope, and defined performance metrics can detect differences in the level of arthroscopic experience. This format of SBI has been deemed acceptable and useful to the intended user, increasing the feasibility of introducing it into surgical training. CLINICAL RELEVANCE: This study adds to the existing body of evidence supporting the potential benefits of benchtop SBIs in arthroscopic skills training. Improved performance on such an SBI may be beneficial for the purpose of basic arthroscopic skills training, and we would support the inclusion of this system in surgical training programs such as those developed by the Arthroscopy Association of North America and American Board of Orthopaedic Surgery.

Elasticity of the coracohumeral ligament in patients with frozen shoulder following rotator interval injection: a case series.

Aim of the study: To evaluate changes in the elasticity of the coracohumeral ligament in patients with adhesive capsulitis of the shoulder treated with ultrasound-guided rotator interval injections. Methods: Shear wave elastography was used to evaluate elasticity of the coracohumeral ligament in symptomatic and asymptomatic shoulders in the shoulder-neutral position and 30° external rotation. A total of 24 shoulders were assessed. Symptomatic shoulders were treated with targeted steroid injection via the rotator interval and manipulation under local anaesthetic block. Follow-up assessment of the elasticity of the coracohumeral ligament was obtained at 10 weeks post-injection. Results: In all subjects, the coracohumeral ligament elastic modulus was larger at 30° external rotation than in the neutral position. In patients with adhesive capsulitis, the coracohumeral ligament thickness and elastic modulus was significantly greater in the symptomatic shoulder in the neutral position and 30° ER. Treated patients had an excellent response with improved Oxford Shoulder Score and reduced visual analogue scale pain scores. Median Oxford Shoulder Score was 13.5 pre-injection and 34 at 10 weeks post-injection. Median visual analogue scale pain scores measured 8.5 pre-injection, 3.5 at 1 day, 2 at 1 week, and 2.5 at 10 weeks. Improved Oxford Shoulder Score and visual analogue scale pain score was associated with a trend to normalisation of the elastic modus of the coracohumeral ligament. Conclusion: In patients with adhesive capsulitis of the shoulder, shear wave elastography demonstrated the coracohumeral ligament is stiffer in the symptomatic shoulder than in the unaffected shoulder. Treatment with the ultrasound-guided rotator interval injection is associated with improved Oxford Shoulder Score, reduced visual analogue scale pain scores, and reduced stiffness in the coracohumeral ligament.

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.