Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition

Metabolic Regulation in Progression to Autoimmune Diabetes

Recent evidence from serum metabolomics indicates that specific metabolic disturbances precede β-cell autoimmunity in humans and can be used to identify those children who subsequently progress to type 1 diabetes. The mechanisms behind these disturbances are unknown. Here we show the specificity of the pre-autoimmune metabolic changes, as indicated by their conservation in a murine model of type 1 diabetes. We performed a study in non-obese prediabetic (NOD) mice which recapitulated the design of the human study and derived the metabolic states from longitudinal lipidomics data. We show that female NOD mice who later progress to autoimmune diabetes exhibit the same lipidomic pattern as prediabetic children. These metabolic changes are accompanied by enhanced glucose-stimulated insulin secretion, normoglycemia, upregulation of insulinotropic amino acids in islets, elevated plasma leptin and adiponectin, and diminished gut microbial diversity of the Clostridium leptum group. Together, the findings indicate that autoimmune diabetes is preceded by a state of increased metabolic demands on the islets resulting in elevated insulin secretion and suggest alternative metabolic related pathways as therapeutic targets to prevent diabetes

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Repair in Trans is kinetically slower than repair in Cis.

<p>(A) Viabilities of the indicated strains (nd indicates not done). Data represent mean ± S.D. (n ≥ 6). (B-D) Kinetics of DSB repair in (B) Cis and Trans configurations, (C) Cis and Reverse-Cis configurations, and (D) Trans and Reverse-Trans configurations as determined by a quantitative PCR assay using primers shown schematically in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g001" target="_blank">Fig 1</a> and listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.s004" target="_blank">S1 Table</a>. The repair of <i>LE</i> and <i>U2</i> ends, which occurs predominantly by synthesis-dependent strand annealing, is indicated in the figure as SDSA, while the repair of the <i>URA3</i> and <i>ura3</i> ends, which occurs by single-strand annealing, is indicated as SSA. The amount of PCR product obtained from a repaired colony was used to make the standard curve for quantification. For Cis and Trans, data represent mean of a total of 6 PCR reactions from two independent time courses ± S.D. For Reverse-Cis and Reverse-Trans, data represent mean of three independent time courses ± S.D.</p

The kinetics of <i>LEU2</i> repair are independent of the repair outcome of another break.

<p>(A) Schematic representation of a modified Cis strain, tNS2607, which carries an <i>LE-HOcs-U2</i> cassette at the <i>can1</i> locus on Chr V, a <i>LEU2</i> donor on Chr III and an unrepairable <i>HOcs-URA3</i> break (instead of a <i>ura3-HOcs-URA3</i> cassette) on Chr XI. (B) Schematic representation of a strain which carries a <i>LE-HOcs-U2</i> cassette at the <i>can1</i> locus on Chr V and a <i>LEU2</i> donor on Chr III (YSJ119 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.ref013" target="_blank">13</a>]). This strain harbors a single HO break. (C) Kinetics of <i>LEU2</i> repair in the indicated strains, as determined by a quantitative PCR assay using primers shown schematically (black arrows) in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g001" target="_blank">1(B)</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g003" target="_blank">3(A) and 3(B)</a>. The amount of PCR product obtained from a repaired colony was used to make the standard curve for quantification. (D) Data from (C) plotted after normalizing the amount of PCR product obtained at 12h time point for each strain to 100%. For Cis and Single Break, data represent mean of a total of 6 PCR reactions from two independent time courses ± S.D. For Modified Cis, data represent mean of three independent time courses ± S.D. The Single Break data shown in (C) has been published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.ref013" target="_blank">13</a>].</p

Adjacent <i>PIR</i> sequences interfere with GC repair in Trans.

<p>(A) Viabilities of the indicated WT, <i>sgs1Δ</i> and <i>msh6Δ</i> 147- and 265- Cis and Trans strains (nd indicates not done). Data represent mean ± S.D. (n ≥ 5). (B) Schematic representation of Chr XI features surrounding the site of insertion of the <i>LE-HOcs-URA3</i> cassette in the 147-Trans strain. The <i>LE-HOcs-URA3</i> cassette was inserted at position 147142 on the left arm of Chr XI. Orange lines represent the <i>PIR</i> genes and the arrowheads indicate their relative orientations. The distance of <i>PIR3</i> gene from <i>PIR1</i> and <i>LE-HOcs-URA3</i> cassette is indicated. The corresponding Cis strain contains a <i>NAT</i>-marked 5’ truncated <i>ura3-HOcs-URA3</i> cassette at position 147172. (C) Rad51 ChIP signal at the <i>LEU2</i> donor on Chr III representing the kinetics of strand-invasion by <i>LE</i> and <i>U2</i> ends in 147- Cis and Trans strains. Primers 300 bp and 200 bp upstream of the <i>LEU2</i> donor and 150 bp and 25 bp downstream of the <i>LEU2</i> donor were used to study the kinetics of strand-invasion by the <i>LE</i> and <i>U2</i> ends, respectively. (D) Schematic representation of the YMV80 SSA strain harboring an <i>HOcs</i> within the <i>leu2</i> gene at its endogenous locus, and a homologous <i>U2</i> sequence at the <i>his4</i> locus ~25 kb distal to the <i>LE-HOcs-U2</i>. Dotted lines indicate the regions spanning the Ty2 retrotransposon element, Ty1 LTRs (long terminal repeats) and tRNA genes that have been deleted in the TyΔ and IRΔ strains, respectively. TyΔ results in a net deletion of 4.7 kb. Figure not drawn to scale.</p

A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair

A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a “recombination execution checkpoint” (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence

Nrf2 and SQSTM1/p62 jointly contribute to mesenchymal transition and invasion in glioblastoma

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM