Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage.

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP-dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP-mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of -0.4. This ∆Lk increases to -0.8 during ATP binding and resets to -0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin-DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a "pinch and merge" mechanism implies that two DNA-binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Depletion or cleavage of cohesin during anaphase differentially affects chromatin structure and segregation.

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.

A Chitosan-based Hydrogel with PLCL, ZnO NPs, and Oligoelements: A Promising Antibiotic Scaffold for Tissue Engineering / Hidrogel de Quitosano con PLCL, ZnO NPs y Oligoelementos: Un Andamio Antibacteriano Prometedor para Ingeniería de Tejidos

ABSTRACT Tissue engineering involves anchorage-dependent cells cultured on scaffolds, with growth factors added to facilitate cell proliferation. Its use in transplants implies the risk of bacterial infection. The current contribution describes the preparation and antibacterial evaluation of a chitosan-based hydrogel physically cross-linked with poly(l-lactic-coɛ-caprolactone) (PLCL) and enriched with zinc oxide nanoparticles (ZnO NPs) and trace elements (potassium and magnesium). The material was developed as a scaffold with built-in antibacterial properties. Chitosan and PLCL are biocompatible support materials applied in medicine for the repair and regeneration of damaged tissues, objectives promoted by ZnO NPs and the aforementioned trace elements. The ZnO NPs were elaborated by chemical coprecipitation. The materials were characterized by XRD, FT-IR, and SEM. Antibacterial testing was performed with strains of Escherichia coli and Staphylococcus aureus by the Kirby-Bauer method, in accordance with the NCCLS and CLSI guidelines. It was possible to obtain a homogeneous hydrogel with adequate morphology and distribution of elements. The hydrogel with 300 mM of Mg, K, and ZnO NP's showed antibacterial inhibition halos of 13 mm for S. aureus and 19 mm for E. coli. This innovative biomaterial with trace elements holds promise for tissue engineering by considering the challenge of bacterial infection.

RESUMEN La ingeniería de tejidos involucra el uso de células cultivadas en andamios con adiciones de factores de crecimiento para facilitar la proliferación celular. Su uso en trasplantes implica riesgo de infección bacteriana. La contribución actual describe la preparación y evaluación antibacteriana de un hidrogel a base de quitosano físicamente reticulado con poli (l-láctico-co-ɛ-caprolactona) (PLCL) enriquecido con nanopartículas de óxido de zinc (NP de ZnO) y oligoelementos (potasio y magnesio). El material se desarrolló como un andamio con propiedades antibacterianas. El quitosano y el PLCL son materiales de soporte biocompatibles aplicados en medicina para la reparación y regeneración de tejidos dañados, propiedades promovidas por las NP´s de ZnO y los oligoelementos antes mencionados. Las NP de ZnO se elaboraron mediante coprecipitación química. Los materiales se caracterizaron por DRX, FT-IR y SEM. Las pruebas antibacterianas se realizaron con cepas de Escherichia coli y Staphylococcus aureus por el método de KirbyBauer de acuerdo con las guías NCCLS y CLSI. Se pudo obtener un hidrogel homogéneo con adecuada morfología y distribución de elementos. El hidrogel con 300 mM de NP ZnO y oligoelementos mostró halos de inhibición antibacteriana de 13 mm para S. aureus y 19 mm para E. coli. Este biomaterial innovador con oligoelementos es prometedor para la ingeniería de tejidos al considerar el desafío de la infección bacteriana.

Leocádia e sua passagem pelos infernos / Leocádia and her journey through hell / Leocádia y su paso por los infiernos

Narrativa que percorre o conjunto de atendimentos clínicos vividos por uma dupla, médica e paciente, no Ambulatório de Arritmia durante o processo de tratamento de uma jovem, sem cardiopatia estrutural, com arritmia ventricular complexa, muito sintomática, sem resposta ao tratamento convencional. Utilizou-se abordagem médica ampliada, não convencional, em que o perfil psicoemocional da paciente foi levado em consideração. Por meio de um método cartográfico, buscou-se delinear os percursos trilhados, ressaltando-se os afetos vividos, os impasses, superações e paradas, enquanto vincos de intensidade que marcaram diversos acontecimentos. A paciente obteve reversão das arritmias e dos sintomas após três anos e meio de tratamento e segue sem arritmia após seis anos e meio. Com isso, acreditamos poder colaborar, de forma construtiva, com o questionamento de diversos aspectos das relações clínicas contemporâneas, das dimensões afetivas do adoecer e de processos implicados na construção da saúde. (AU)

arrative depicting the clinical care journey experienced by a doctor and her patient-a young woman with symptomatic ventricular tachycardia, with no structural heart disease, with no response to conventional treatment-at the Arrhythmia Outpatient Clinic. The non-conventional expanded medical approach-where the patient's psychoemotional profile is taken into consideration - was adopted. Through a cartographic method, we aim to trace the paths taken, highlighting feelings, deadlocks, achievements, and stoppage moments as lines of intensity marking several events. The patient reversed her arrhythmia and symptoms after three and a half years of treatment, and remains so after six and a half years. Therefore, we believe we can constructively cooperate with the discussions of several aspects of contemporary clinical relations, affective dimensions of becoming ill, and processes implied in the development of health. (AU)

Narrativa que trascurre por el conjunto de atenciones clínicas vividas por dos personas, una médica y una paciente, en el Ambulatorio de Arritmia, durante el proceso de tratamiento de una joven, sin cardiopatía estructural, con arritmia ventricular compleja, muy sintomática, sin respuesta al tratamiento convencional. Se utilizó el abordaje médico ampliado, no convencional, en el que el perfil psicoemocional de la paciente se llevó en consideración. Por medio de un método cartográfico, se buscó delinear los recorridos seguidos, subrayándose los afectos vividos, los callejones sin salida, las superaciones y las paradas, como marcas de intensidad que señalaron diversos acontecimientos. La paciente tuvo reversión de las arritmias y de los síntomas después de tres años y medio de tratamiento y continúa sin arritmia pasados seis años y medio. De esa forma, creemos que podemos colaborar, de forma constructiva, con el cuestionamiento de diversos aspectos de las relaciones clínicas contemporáneas, de las dimensiones afectivas del enfermarse y de procesos implicados en la construcción de la salud. (AU)

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction.

Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.

Cryo-EM structures of holo condensin reveal a subunit flip-flop mechanism.

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.

Sumoylation of Smc5 Promotes Error-free Bypass at Damaged Replication Forks.

Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.

A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules.

Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a "permanent bridge" resisting forces over 80 pN and a force-sensitive "reversible bridge." The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. "Permanent" cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.

FACT mediates cohesin function on chromatin.

Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.

Synthetic studies on the reverse antibiotic natural products, the nybomycins.

Antimicrobial resistance (AMR) is a serious issue that could have severe consequences if steps are not taken. The nybomycin natural products have the potential to extend the clinical efficacy of the marketed fluoroquinolone class of antibiotics through a 'reverse antibiotic' approach. However, only very limited structure-activity relationships are known for these fascinating compounds, in part due to challenges with their synthesis. Here we report a new scalable and robust synthetic route to the nybomycin natural products to aid in the development of this series. Through this synthesis, we report the antibiotic activity of novel analogues of this family confirming the selectivity for fluoroquinolone resistant bacteria and potential future opportunities for further optimisation.

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection.

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.

The Smc5/6 Complex: New and Old Functions of the Enigmatic Long-Distance Relative.

Smc5 and Smc6, together with the kleisin Nse4, form the heart of the enigmatic and poorly understood Smc5/6 complex, which is frequently viewed as a cousin of cohesin and condensin with functions in DNA repair. As novel functions for cohesin and condensin complexes in the organization of long-range chromatin architecture have recently emerged, new unsuspected roles for Smc5/6 have also surfaced. Here, I aim to provide a comprehensive overview of our current knowledge of the Smc5/6 complex, including its long-established function in genome stability, its multiple roles in DNA repair, and its recently discovered connection to the transcription inhibition of hepatitis B virus genomes. In addition, I summarize new research that is beginning to tease out the molecular details of Smc5/6 structure and function, knowledge that will illuminate the nuclear activities of Smc5/6 in the stability and dynamics of eukaryotic genomes.

Identification of SUMO conjugation sites in the budding yeast proteome.

Post-translational modification by the small ubiquitin-like modifier (SUMO) is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

Hydrological modeling of the Peruvian-Ecuadorian Amazon basin using GPM-IMERG satellite-based precipitation dataset.

In the last two decades, rainfall estimates provided by the Tropical Rainfall Measurement Mission (TRMM) have proven applicable in hydrological studies. The Global Precipitation Measurement (GPM) mission, which provides the new generation of rainfall estimates, is now considered a global successor to TRMM. The usefulness of GPM data in hydrological applications, however, has not yet been evaluated over the Andean and Amazonian regions. This study uses GPM data provided by the Integrated Multi-satellite Retrievals (IMERG) (product/final run) as input to a distributed hydrological model for the Amazon Basin of Peru and Ecuador for a 16-month period (from March 2014 to June 2015) when all datasets are available. TRMM products (TMPA V7, TMPA RT datasets) and a gridded precipitation dataset processed from observed rainfall are used for comparison. The results indicate that precipitation data derived from GPM-IMERG correspond more closely to TMPA V7 than TMPA RT datasets, but both GPM-IMERG and TMPA V7 precipitation data tend to overestimate, compared to observed rainfall (by 11.1% and 15.7 %, respectively). In general, GPM-IMERG, TMPA V7 and TMPA RT correlate with observed rainfall, with a similar number of rain events correctly detected (~20%). Statistical analysis of modeled streamflows indicates that GPM-IMERG is as useful as TMPA V7 or TMPA RT datasets in southern regions (Ucayali basin). GPM-IMERG, TMPA V7 and TMPA RT do not properly simulate streamflows in northern regions (Marañón and Napo basins), probably because of the lack of adequate rainfall estimates in northern Peru and the Ecuadorian Amazon.

Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions.

Chromosome condensation is an essential morphological event required for successful DNA segregation during mitosis. The high level of genome compaction achieved during this process is attained by the evolutionary conserved condensin complex. Recently, several lines of evidences have demonstrated that the mitotic phosphatase Cdc14 is required to ensure condensin loading onto chromosomes. To date several approaches have been used in order to characterize condensin activity and regulation, however these techniques are time-consuming and require complex equipment. In this chapter we described an easy and reliable protocol to analyze Cdc14-dependent condensin loading onto specific genomic DNA regions by using a chromatin immunoprecipitation (ChIP) technique.