RESUMO
Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Coesinas , Segregação de Cromossomos , Mutação , Cromátides/metabolismo , Cromátides/genética , Evolução Molecular , Meiose/genéticaRESUMO
Ribosomal RNA genes (rDNAs) are located in large domains of hundreds of rDNA units organized in a head-to-tail manner. The proper and stable inheritance of rDNA clusters is of paramount importance for survival. Yet, these highly repetitive elements pose a potential risk to the genome since they can undergo non-allelic exchanges. Here, we review the current knowledge of the organization of the rDNA clusters in Arabidopsis thaliana and their stability during meiosis. Recent findings suggest that during meiosis, all rDNA loci are embedded within the nucleolus favoring non-homologous end joining (NHEJ) as a repair mechanism, while DNA repair via homologous recombination (HR) appears to be a rare event. We propose a model where (1) frequent meiotic NHEJ events generate abundant single nucleotide polymorphisms and insertions/deletions within the rDNA, resulting in a heterogeneous population of rDNA units and (2) rare HR events dynamically change rDNA unit numbers, only to be observed in large populations over many generations. Based on the latest efforts to delineate the entire rDNA sequence in A. thaliana, we discuss evidence supporting this model. The results compiled so far draw a surprising picture of rDNA sequence heterogeneity between individual units. Furthermore, rDNA cluster sizes have been recognized as relatively stable when observing less than 10 generations, yet emerged as major determinant of genome size variation between different A. thaliana ecotypes. The sequencing efforts also revealed that transcripts from the diverse rDNA units yield heterogenous ribosome populations with potential functional implications. These findings strongly motivate further research to understand the mechanisms that maintain the metastable state of rDNA loci.
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In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Proteínas de Arabidopsis/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Recombinação GenéticaRESUMO
Visualization of meiotic chromosomes and the proteins involved in meiotic recombination have become essential to study meiosis in many systems including the model plant Arabidopsis thaliana. Recent advances in super-resolution technologies changed how microscopic images are acquired and analyzed. New technologies enable observation of cells and nuclei at a nanometer scale and hold great promise to the field since they allow observing complex meiotic molecular processes with unprecedented detail. Here, we provide an overview of classical and advanced sample preparation and microscopy techniques with an updated Arabidopsis meiotic atlas based on super-resolution microscopy. We review different techniques, focusing on stimulated emission depletion (STED) nanoscopy, to offer researchers guidance for selecting the optimal protocol and equipment to address their scientific question.
RESUMO
Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.
Assuntos
Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , Meiose , Recombinação Genética/genética , Complexo Sinaptonêmico/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cromatina/metabolismo , Troca Genética , Reparo do DNA , Fertilidade , Mutação/genética , Recombinases/metabolismoRESUMO
Despite vast differences between organisms, some characteristics of their genomes are conserved, such as the nucleolus organizing region (NOR). The NOR is constituted of multiple, highly repetitive rDNA genes, encoding the catalytic ribosomal core RNAs which are transcribed from 45S rDNA units. Their precise sequence information and organization remain uncharacterized. Here, using a combination of long- and short-read sequencing technologies we assemble contigs of the Arabidopsis NOR2 rDNA domain. We identify several expressed rRNA gene variants which are integrated into translating ribosomes in a tissue-specific manner. These findings support the concept of tissue specific ribosome subpopulations that differ in their rRNA composition and provide insights into the higher order organization of NOR2.
Assuntos
Arabidopsis/genética , DNA de Plantas/genética , DNA Ribossômico/genética , Genoma de Planta/genética , Região Organizadora do Nucléolo/genética , Cromossomos Artificiais Bacterianos/genética , Mapeamento de Sequências Contíguas , Biblioteca Gênica , RNA Ribossômico/genética , Ribossomos/genética , Análise de Sequência de DNARESUMO
Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Quinases Ciclina-Dependentes/genética , Proteínas de Ligação a DNA/química , Mutação , Fosforilação , Ligação Proteica , Multimerização ProteicaRESUMO
Visualization of meiotic chromatin from pollen mother cells has become an essential technique to study meiosis in the model plant Arabidopsis thaliana. Here we present an advanced cytogenetic method that combines improved immunocytology with chromosome painting, thereby generating a tool to quantitatively analyze localization of proteins to any given genomic region. Proteins involved in different processes such as DNA double-strand break formation and recombinational repair can be visualized on meiotic chromatin with the additional feature of assessing their abundance at specific chromosomal locations.
Assuntos
Cromatina/genética , Coloração Cromossômica , Meiose , Arabidopsis/genética , Cromatina/metabolismo , Cromossomos de Plantas , Análise Citogenética/métodos , Hibridização in Situ FluorescenteRESUMO
Imaging cells, nuclei, and DNA in their natural spatial contexts and configurations is challenging yet required to understand the biology of genome organization, maintenance, and transmission. Live-cell imaging allows capturing dynamic changes of chromosomes in their nuclear and cellular context but lacks resolution. In contrast, imaging of fixed, spread chromosome samples provides unmatched resolution but potentially distorts configurations and spatial relations. Fixed whole-mount samples preserve chromosome configurations and cellular contexts and allow high-resolution imaging. Importantly the latter method allows simultaneous visualization of specific genomic regions (via fluorescent in situ hybridization-FISH) and proteins (via immune-localization using antibodies or tags). Here we present an advanced "whole-mount immuno-FISH" (WhoMI-FISH) method based on the published protocol by Bey Till et al. (Methods Mol Biol 1675:467-480, 2018) specifically optimized for pollen mother cells (PMCs) of Arabidopsis thaliana. It focuses on (1) specimen preparation that maintains meiocyte nuclei positions and genome organization in anthers and also on (2) simultaneous detection of specific genomic regions and meiotic proteins.
Assuntos
Arabidopsis/genética , Hibridização in Situ Fluorescente , Meiose , Cromossomos de Plantas , Hibridização in Situ Fluorescente/métodosRESUMO
Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo nonallelic exchanges. During meiosis, DNA double-strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR), which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis (Arabidopsis thaliana), we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications and meiotic cohesin and allows only very limited meiosis-specific DSB formation. DNA lesions within the rDNA arrays are repaired in an RAD51-independent but LIG4-dependent manner, establishing that nonhomologous end-joining maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats, we validate our findings and demonstrate that the rDNA constitutes an HR-refractory genome environment.
Assuntos
Arabidopsis/genética , Nucléolo Celular , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/fisiologia , Meiose/fisiologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , DNA Ligases/metabolismo , DNA de Plantas , DNA Ribossômico/genética , Instabilidade Genômica , Recombinação Homóloga , Plantas Geneticamente Modificadas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , CoesinasRESUMO
In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Hidrólise , Rad51 Recombinase/isolamento & purificação , Recombinases Rec A/isolamento & purificação , Alinhamento de SequênciaRESUMO
In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0017-6, 2018) and a Reply by the authors of the Protocol (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0018-5, 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced.
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This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.
Assuntos
Previsões/métodos , Espectrometria de Massas/métodos , Estrutura Terciária de Proteína/fisiologia , Simulação por Computador , Reagentes de Ligações Cruzadas/química , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Proteínas/genética , Proteínas/fisiologiaRESUMO
Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reparo do DNA/genética , DNA de Plantas/genética , Recombinação Homóloga/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Meiose/genética , MutaçãoRESUMO
DNA double-strand break (DSB) repair depends on the ataxia telangiectasia mutated (ATM) kinase that phosphorylates the conserved C-terminal SQ motif present in the histone variant H2A.X [1-7]. In constitutive heterochromatin of mammals, DSB repair is delayed and relies on phosphorylation of the proteins HP1 and KAP1 by ATM [2, 8-14]. However, KAP1 is not conserved in plants and the HP1-related protein Like-HP1 (LHP1) is not localized at constitutive heterochromatin [15], suggesting that in plants, alternative mechanisms could be responsible for repair of DSBs in heterochromatin. In Arabidopsis, constitutive heterochromatin is marked by H3K9 methylation and the plant-specific histone variants H2A.W, which are distinguished by their C-terminal motif KSPKK and required for heterochromatin compaction [16-18]. We report that the Arabidopsis histone variant H2A.W.7 is confined to heterochromatin and carries a SQ motif that is phosphorylated by ATM. In response to DNA damage, phosphorylation of H2A.W.7 takes place in heterochromatin, while H2A.X phosphorylation takes place primarily in euchromatin. We propose that H2A.W.7 evolved in addition to H2A.X to facilitate DNA damage response in highly condensed heterochromatin, thus playing a role similar to KAP1 and HP1 phosphorylation in mammals. These data support the idea of the functional diversification of histone variants and their role in spatial compartmentalization of chromatin-related functions in eukaryotes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dano ao DNA , Histonas/metabolismo , Mutação , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Reparo do DNA , Eucromatina , Heterocromatina , Histonas/genética , FosforilaçãoRESUMO
The HOP2-MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2-MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL-MS). The final XL-MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC-MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS(2)G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2-MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution.
Assuntos
Proteínas de Arabidopsis/química , Fosfotransferases/química , Proteínas de Arabidopsis/metabolismo , Carbodi-Imidas/química , Cromatografia em Gel , Reagentes de Ligações Cruzadas/química , Modelos Moleculares , Complexos Multiproteicos/química , Fosfotransferases/metabolismo , Conformação Proteica , Multimerização Proteica , Succinimidas/química , Espectrometria de Massas em Tandem , Fluxo de TrabalhoRESUMO
The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).
Assuntos
Arabidopsis/crescimento & desenvolvimento , Proteínas Mutadas de Ataxia Telangiectasia/genética , Espectrometria de Massas/métodos , Fosfoproteínas/isolamento & purificação , Proteômica/métodos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , MutaçãoRESUMO
Meiosis is a specialized cell division in sexually reproducing organisms before gamete formation. Following DNA replication, the canonical sequence in species with monocentric chromosomes is characterized by reductional segregation of homologous chromosomes during the first and equational segregation of sister chromatids during the second meiotic division. Species with holocentric chromosomes employ specific adaptations to ensure regular disjunction during meiosis. Here we present the analysis of two closely related plant species with holocentric chromosomes that display an inversion of the canonical meiotic sequence, with the equational division preceding the reductional. In-depth analysis of the meiotic divisions of Rhynchospora pubera and R. tenuis reveals that during meiosis I sister chromatids are bi-oriented, display amphitelic attachment to the spindle and are subsequently separated. During prophase II, chromatids are connected by thin chromatin threads that appear instrumental for the regular disjunction of homologous non-sister chromatids in meiosis II.