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1.
Proc Natl Acad Sci U S A ; 119(19): e2203967119, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35503911

RESUMO

SignificanceH-DNA is a non-B-form DNA structure containing intramolecular triplex and single-stranded DNA (ssDNA) regions. H-DNA-forming motifs include polypyrimidine•polypurine mirror repeat sequences, which occur frequently in eukaryotic genomes. These motifs have biological impacts on genome stability and processes such as replication and transcription, but their non-B DNA-forming potentials are not fully understood. Here, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated by a deep-sequencing technology that uses the ssDNA-specific nuclease S1 to detect ssDNA. It is currently unclear whether the H-DNA detected formed in vitro or was already present in vivo. Nevertheless, this study provides an approach to unveiling structural features of intramolecular triplexes genome-wide at high spatial resolution.


Assuntos
Genoma , Motivos de Nucleotídeos , Animais , Sequência de Bases , Genoma/genética , Camundongos , Conformação de Ácido Nucleico
2.
Cureus ; 14(1): e21668, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35237471

RESUMO

Unlike the tick-borne diseases ehrlichiosis and Lyme disease, human granulocytic anaplasmosis is rarely associated with neurological complications. In this case report, we present a patient who developed a severe, lancinating headache shortly after known tick exposure. A tick-borne PCR panel was positive for Anaplasmosis phagocytophilum and neurology evaluation yielded a concomitant diagnosis of new-onset trigeminal neuralgia. Our case explores the relationship between anaplasmosis infection and trigeminal neuralgia.

3.
BMC Endocr Disord ; 22(1): 69, 2022 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-35296307

RESUMO

BACKGROUND: Diabetes mellitus affects 13% of American adults. To address the complex care requirements necessary to avoid diabetes-related morbidity, the American Diabetes Association recommends utilization of multidisciplinary teams. Research shows pharmacists have a positive impact on multiple clinical diabetic outcomes. METHODS: Open-label randomized controlled trial with 1:1 assignment that took place in a single institution resident-run outpatient medicine clinic. Patients 18-75 years old with type 2 diabetes mellitus and most recent HbA1c ≥9% were randomized to standard of care (SOC) (continued with routine follow up with their primary provider) or to the SOC + pharmacist-managed diabetes clinic PMDC group (had an additional 6 visits with the pharmacist within 6 months from enrollment). Patients were followed for 12 months after enrollment. Data collected included HbA1c, lipid panel, statin use, blood pressure control, immunization status, and evidence of diabetic complications (retinopathy, nephropathy, neuropathy). Intention-to-treat and per-protocol analysis were performed. RESULTS: Forty-four patients were enrolled in the SOC + PMDC group and 42 patients in the SOC group. Average decrease in HbA1c for the intervention compared to the control group at 6 months was - 2.85% vs. -1.32%, (p = 0.0051). Additionally, the odds of achieving a goal HbA1c of ≤8% at 6 months was 3.15 (95% CI = 1.18, 8.42, p = 0.0222) in the intervention versus control group. There was no statistically significant difference in the remaining secondary outcomes measured. CONCLUSIONS: Addition of pharmacist managed care for patients with type 2 diabetes mellitus is associated with significant improvements in HbA1c compared with standard of care alone. Missing data during follow up limited the power of secondary outcomes analyses. TRIAL REGISTRATION: ClinicalTrials.gov , ID: NCT03377127 ; first posted on 19/12/2017.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação de Processos e Resultados em Cuidados de Saúde , Ambulatório Hospitalar/organização & administração , Farmacêuticos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
4.
Genes Dev ; 36(3-4): 180-194, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058317

RESUMO

Mechanisms regulating meiotic progression in mammals are poorly understood. The N 6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.


Assuntos
Meiose , RNA Helicases , Animais , Regulação da Expressão Gênica , Masculino , Mamíferos/genética , Meiose/genética , Camundongos , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética
5.
Cell Rep ; 37(11): 110110, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34910909

RESUMO

Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2 is highly expressed in pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with m6A status, and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets mRNAs for degradation. Furthermore, altered transcripts in mutant pachytene cells encode microtubule network proteins. Our results demonstrate that YTHDC2 regulates the pachytene stage by perpetuating a meiotic transcriptome and preventing microtubule network changes that could lead to telomere clustering.


Assuntos
Meiose , Microtúbulos/fisiologia , Estágio Paquíteno , RNA Helicases/fisiologia , Espermatócitos/citologia , Telômero , Transcriptoma , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatócitos/metabolismo
7.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
8.
Curr Biol ; 31(21): R1442-R1444, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34752773

RESUMO

Meiosis depends on the cell's ability to match each chromosome to its homolog in a strictly pairwise fashion. A new study describes an elegant mechanism that tetraploid Arabidopsis arenosa plants evolved to faithfully connect and segregate pairs of homologous chromosomes.


Assuntos
Arabidopsis , Meiose , Arabidopsis/genética , Cromossomos de Plantas/genética , Poliploidia , Tetraploidia
9.
Cell ; 184(24): 5970-5984.e18, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34793701

RESUMO

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.


Assuntos
Deleção de Genes , Duplicação Gênica , Células Germinativas/metabolismo , Recombinação Genética/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Cromátides/metabolismo , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Quebras de DNA de Cadeia Dupla , DNA Circular/genética , Feminino , Genoma , Haplótipos/genética , Recombinação Homóloga/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutagênese Insercional/genética , Mutação/genética
10.
Nature ; 594(7864): 572-576, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34108687

RESUMO

Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots3,4, yet how Spo11 engages with its DNA substrate to catalyse DNA cleavage is poorly understood. Although most recombination events are initiated by a single Spo11 cut, here we show in Saccharomyces cerevisiae that hyperlocalized, concerted Spo11 DSBs separated by 33 to more than 100 base pairs also form, which we term 'double cuts'. Notably, the lengths of double cuts vary with a periodicity of 10.5 base pairs, which is conserved in yeast and mice. This finding suggests a model in which the orientation of adjacent Spo11 molecules is fixed relative to the DNA helix-a proposal supported by the in vitro DNA-binding properties of the Spo11 core complex. Deep sequencing of meiotic progeny identifies recombination scars that are consistent with repair initiated from gaps generated by adjacent Spo11 DSBs. Collectively, these results revise our present understanding of the mechanics of Spo11-DSB formation and expand on the original concepts of gap repair during meiosis to include DNA gaps that are generated by Spo11 itself.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Meiose , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animais , Reparo do DNA , Camundongos , Camundongos Knockout
11.
Nature ; 592(7852): 144-149, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33731927

RESUMO

The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein1,2. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures3-5, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery2. Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Nucleoproteínas/metabolismo , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , DNA Fúngico/química , Endodesoxirribonucleases/metabolismo , Recombinação Homóloga , Proteínas Nucleares/química , Nucleoproteínas/química , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Recombinases/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
12.
PLoS Genet ; 17(2): e1009265, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33635934

RESUMO

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5' ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development.


Assuntos
Infertilidade Masculina/genética , Meiose/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/metabolismo , Retroelementos/genética , Espermatogênese/genética , Alelos , Animais , Inativação Gênica , Células Germinativas/metabolismo , Células Germinativas/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas Mitocondriais/genética , Mutação , Estágio Paquíteno/genética , Fosfolipase D/genética , RNA Helicases/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Testículo/metabolismo
13.
Curr Genet ; 67(3): 431-437, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33604699

RESUMO

The programmed formation of DNA double-strand breaks (DSBs) in meiotic prophase I initiates the homologous recombination process that yields crossovers between homologous chromosomes, a prerequisite to accurately segregating chromosomes during meiosis I (MI). In the budding yeast Saccharomyces cerevisiae, proteins required for meiotic DSB formation (DSB proteins) accumulate to higher levels specifically on short chromosomes to ensure that these chromosomes make DSBs. We previously demonstrated that as-yet undefined cis-acting elements preferentially recruit DSB proteins and promote higher levels of DSBs and recombination and that these intrinsic features are subject to selection pressure to maintain the hyperrecombinogenic properties of short chromosomes. Thus, this targeted boosting of DSB protein binding may be an evolutionarily recurrent strategy to mitigate the risk of meiotic mis-segregation caused by karyotypic constraints. However, the underlining mechanisms are still elusive. Here, we discuss possible scenarios in which components of the meiotic chromosome axis (Red1 and Hop1) bind to intrinsic features independent of the meiosis-specific cohesin subunit Rec8 and DNA replication, promoting preferential binding of DSB proteins to short chromosomes. We also propose a model where chromosome position in the nucleus, influenced by centromeres, promotes the short-chromosome boost of DSB proteins.


Assuntos
Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Prófase Meiótica I/genética , Saccharomyces cerevisiae/genética
14.
Nat Struct Mol Biol ; 28(1): 92-102, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398171

RESUMO

Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Microscopia de Força Atômica , Mutação/genética , Conformação de Ácido Nucleico , Recombinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Genes Dev ; 34(23-24): 1605-1618, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33184224

RESUMO

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.


Assuntos
Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Retroalimentação Fisiológica/fisiologia , Meiose/genética , Saccharomyces cerevisiae/genética , Complexo Sinaptonêmico/genética , Aneuploidia , Pareamento Cromossômico/genética , Recombinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
16.
Proc Natl Acad Sci U S A ; 117(48): 30577-30588, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199619

RESUMO

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Troca Genética , Exodesoxirribonucleases/metabolismo , Meiose/genética , Proteínas MutL/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cromossomos Fúngicos , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinação Genética
17.
Mol Cell ; 79(4): 689-701.e10, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32610038

RESUMO

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Meiose , Proteínas de Ligação a Fosfato/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Troca Genética , DNA de Cadeia Simples/metabolismo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Testículo
18.
BMC Cardiovasc Disord ; 20(1): 263, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487114

RESUMO

BACKGROUND: The benefit of combining aspirin and direct oral anticoagulants on the reduction of cardiovascular events in atrial fibrillation or flutter is not well studied. We aimed to assess whether concurrent aspirin and direct oral anticoagulant therapy for atrial fibrillation or flutter will result in less coronary, cerebrovascular and systemic ischemic events compared to direct oral anticoagulant therapy alone. METHODS: Retrospective study of adult patients between 18 and 100 years old who have nonvalvular atrial fibrillation or flutter and were started on a direct oral anticoagulant (apixaban, rivaroxaban, or dabigatran), between January 1, 2010 and September 1, 2015 within the Beaumont Health System. Exclusions were history of venous thromboembolic disease and use of other antiplatelet therapies such as P2Y12 inhibitors. Patients were classified into two groups based on concurrent aspirin use and observed for a minimum of 2 years. Primary outcome was major adverse cardiac events, defined as acute coronary syndromes, ischemic strokes, and embolic events. Secondary outcomes were bleeding and death. RESULTS: Six thousand four patients were in the final analysis, 57% males and 80% Caucasians, median age 71, interquartile range (63-80). The group exposed to aspirin contained 2908 subjects, and the group unexposed to aspirin contained 3096 subjects. After using propensity scores to balance the baseline characteristics in both groups, the analysis revealed higher rate of major adverse cardiac events in the exposed group compared to the unexposed group, (HR 2.11, 95% CI (1.74-2.56)) with a number needed to harm of 11 (95% CI [9-11]). The rate of bleeding was also higher in the exposed group, (HR 1.30, 95% CI (1.11-1.52)). The rate of death was not statistically different between the groups, (HR 0.87, 95% CI (0.61-1.25)). CONCLUSIONS: In this observational analysis of patients with atrial fibrillation and flutter, the concomitant use of direct oral anticoagulants and aspirin was associated with an increased risk of both major adverse cardiac and bleeding events when compared to the use of direct oral anticoagulants alone. These findings underscore the potential harm of this combination therapy when used without a clear indication.


Assuntos
Síndrome Coronariana Aguda/prevenção & controle , Aspirina/administração & dosagem , Fibrilação Atrial/tratamento farmacológico , Flutter Atrial/tratamento farmacológico , Embolia/prevenção & controle , Inibidores do Fator Xa/administração & dosagem , AVC Isquêmico/prevenção & controle , Inibidores da Agregação Plaquetária/administração & dosagem , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/mortalidade , Administração Oral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspirina/efeitos adversos , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/mortalidade , Flutter Atrial/diagnóstico , Flutter Atrial/mortalidade , Embolia/diagnóstico , Embolia/mortalidade , Inibidores do Fator Xa/efeitos adversos , Feminino , Hemorragia/induzido quimicamente , Humanos , AVC Isquêmico/diagnóstico , AVC Isquêmico/mortalidade , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/efeitos adversos , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
19.
Nature ; 582(7810): 124-128, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494071

RESUMO

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.


Assuntos
Cromossomos Fúngicos/genética , Recombinação Homóloga , Meiose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Período de Replicação do DNA , Meiose/genética , Prófase Meiótica I/genética , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Fatores de Tempo
20.
Mol Cell ; 78(6): 1252-1263.e3, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32362315

RESUMO

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.


Assuntos
Troca Genética/fisiologia , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Animais , Segregação de Cromossomos/genética , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos DBA , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ácidos Nucleicos Heteroduplexes/genética
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