The Recombinases Rad51 and Dmc1 Play Distinct Roles in DNA Break Repair and Recombination Partner Choice in the Meiosis of Tetrahymena

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3′ single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA–strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB–dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk

A Single Cohesin Complex Performs Mitotic and Meiotic Functions in the Protist <i>Tetrahymena</i>

<div><p>The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist <i>Tetrahymena thermophila</i>. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, <i>Tetrahymena</i> uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.</p> </div

Localization of Rec8p, Smc1p, and Scc3p to mitotic and meiotic chromosomes.

<p>(A, B) Rec8-GFP (red) and Smc1-HA (yellow) localize to interphase and mitotic MICs. (C) Rec8p localizes to MIC chromosomes during all stages of meiosis (both mating partners express GFP-tagged Rec8). (D) Smc1p localizes to MIC chromosomes during all stages of meiosis (only one partner expresses HA-tagged Smc1). Note the absence of both cohesion proteins from the MACs. (E) The Scc3p homolog TTHERM_00225630p (green) localizes to mitotic and meiotic MICs of all stages. (F) Paired cells in meiotic anaphase II prepared by a spreading method to remove free nuclear proteins. Persistance of Rec8 staining (red) demonstrates that it remains attached to the arms of separating chromatids. (G) MAC localization of Rec8p is not increased, whereas repair protein Rad51 is strongly expressed in the MAC upon DNA damage. Bar: 10 µm in A–F.</p

Phenotypes of cohesin-depleted cells.

<p>(A) Rec8p is depleted by cadmium (Cd)-induced expression of hairpin RNA and monitored by the detection of GFP-tagged Rec8p on a Western gel. RNAi for 24 h leads to virtually complete loss of Rec8p. GFP band intensities were determined using the “Analyze” tool of ImageJ (Wayne Rasband, N.I.H.; <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>), normalized to the α-tubulin loading control, and used to calculate relative protein amounts. (B) RT-PCR shows that <i>REC8</i> RNA is greatly reduced after 2 h of RNAi induction, and nearly absent after 24 h. (C) Mitotic division of the MIC shows delayed segregation and lagging chromosomes, and the MAC also splits abnormally in cells depleted of Rec8p and Smc1p. Bar: 10 µm. (D) Depletion of Rec8p causes the loss of Smc1p in mitotic and meiotic MICs. In the presence of Rec8p, Smc1-HA localizes to MICs in vegetatively propagating cells, but disappears when <i>rec8</i> RNAi is induced. When REC8hp/Smc1-HA cells are mated to a wild type (WT) partner, the former show micronuclear Smc1-HA localization, which disappears upon <i>rec8</i> RNAi. Bar: 10 µm. (E) <i>rec8</i>i cells (top) arrest at an abnormal metaphase-anaphase I stage, whereas the WT mating partners (bottom) show the normal progression of meiosis. In WT metaphase, 5 distinct bivalents can be seen (arrow points to one well-separated bivalent), whereas the <i>rec8</i>i partner shows fragmented chromosomes. Bar: 10 µm. (F) Quantitation of <i>rec8</i>i meiosis arrest 4,5 h after meiosis induction. Eliminating DSBs by mating <i>rec8</i>i cells with <i>spo11</i>i partners rescues the <i>rec8</i>i phenotype. Stages of <i>rec8</i>i cells were scored in cell pairs where their respective WT or <i>spo11</i>i mating partners had progressed beyond the stage indicated (n = 100 cells in both experiments). (G) DSB markers g-H2AX (orange) and Dmc1p (green) highlight elongated meiotic prophase MICs (left) in both <i>rec8</i>i and WT cells of a mating pair. Later in meiosis (right), when they have disappeared from the WT partner, they are still present in the arrested MICs of the <i>rec8</i>i partner (arrows). (H) Detection of DSBs by PFGE in WT and <i>rec8</i>i meiosis. The lower panel shows a control hybridization to the same membrane as a test for equal DNA loading and Southern transfer. (I) Evaluation of pairing (scored as the presence of a single FISH signal) and loss of cohesion (scored as the presence of 3 or 4 signals) in elongated meiotic prophase MICs, displayed by different genotypes. Examples of MICs with different numbers of FISH signals are shown on top. Values are means of three repeats with 50 nuclei evaluated, each. Error bars indicate standard deviation.</p

RNAi depletion of separase Esp1p prevents mitotic and meiotic division and causes MIC polyploidization.

<p>(A) Chromosomes fail to segregate during mitosis. Whereas in the WT MIC, division precedes the splitting of the MAC, the <i>esp1</i>i MIC unsuccessfully attempts division and eventually remains as a single unreduced MIC in one of the two daughter cells. Staining for phosH3(Ser10) (green) highlights only condensed mitotic chromosomes in the WT, while undivided <i>esp1i</i> nuclei remain in a permanently condensed state. (B) After 48 hours of <i>esp1</i>i induction, MICs become very large and contain ropy structures, suggesting bundles of unseparated chromatids. (C) FISH with a probe against a unique chromosomal locus shows that, whereas the WT MIC contains two copies of the locus, <i>esp1</i>i MICs are polyploid. Unlike in the MAC, multiple copies of the locus are not dispersed, but form clusters. This suggests that replicated chromatids do not fall apart but remain in bundles resembling polytenic chromosomes. (D) FACScan analysis after 48 hours of <i>esp1</i>i induction confirms that MICs become polyploid due to failing segregation. While MICs and MACs form peaks of different DNA contents in the WT, in the <i>esp1</i>i sample they coalesce into a single peak roughly equivalent to the WT MAC DNA content. (E) Meiotic prophase stages are normal but bivalents fail to separate at anaphase I and coalesce back to a single MIC in the absence of Esp1p (compare with the WT situation in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003418#pgen-1003418-g001" target="_blank">Figure 1B</a>). (F) Quantitation of the <i>esp1</i>i meiotic arrest 5,5 h after meiosis induction. Bar in (A) represents 10 µm in (A) and (E), bar in (B) represents 10 µm in (B) and (C).</p