RESUMEN
Telomere-led rapid chromosome movements (RPMs) are a conserved characteristic of chromosome dynamics in meiosis. RPMs have been suggested to influence critical meiotic functions such as DNA repair and the association of the homologous chromosomes. Here, we describe a method using 3D time-lapse fluorescence imaging to monitor RPMs in Hoechst-stained mouse seminiferous tubules explants. We supplement visualization with customized quantitative motion analysis and in silico simulation. The ability to carry out live imaging, combined with quantitative image analysis, offers a sensitive tool to investigate the regulation of RPMs, chromosome reorganizations that precede dynamic mid-prophase events, and their contribution to faithful transmission of genetic information.
Asunto(s)
Meiosis , Animales , Ratones , Masculino , Imagen de Lapso de Tiempo/métodos , Telómero/genética , Telómero/metabolismo , Túbulos Seminíferos/citología , Túbulos Seminíferos/metabolismo , Cromosomas/genéticaRESUMEN
Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Espermatocitos , Animales , Espermatocitos/metabolismo , Espermatocitos/citología , Ratones , Masculino , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Meiosis/genética , Inmunoprecipitación de Cromatina/métodos , Sitios de UniónRESUMEN
Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Factores de Transcripción , Animales , Aurora Quinasa B/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Meiosis/genética , Ratones , Factores de Transcripción/metabolismoRESUMEN
Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.
Asunto(s)
ADN Helicasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Espermatogonias , Animales , Diferenciación Celular , Gónadas , Masculino , Mamíferos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Factores de Transcripción/genéticaRESUMEN
Telomere-led rapid chromosome movements or rapid prophase movements direct fundamental meiotic processes required for successful haploidization of the genome. Critical components of the machinery that generates rapid prophase movements are unknown, and the mechanism underlying rapid prophase movements remains poorly understood. We identified S. cerevisiae Mps2 as the outer nuclear membrane protein that connects the LINC complex with the cytoskeleton. We also demonstrate that the motor Myo2 works together with Mps2 to couple the telomeres to the actin cytoskeleton. Further, we show that Csm4 interacts with Mps2 and is required for perinuclear localization of Myo2, implicating Csm4 as a regulator of the Mps2-Myo2 interaction. We propose a model in which the newly identified functions of Mps2 and Myo2 cooperate with Csm4 to drive chromosome movements in meiotic prophase by coupling telomeres to the actin cytoskeleton.
Asunto(s)
Cromosomas Fúngicos/fisiología , Proteínas de la Membrana/genética , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas Nucleares/genética , Profase/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Citoesqueleto de Actina/fisiología , Citoesqueleto/fisiología , Meiosis/fisiología , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/fisiologíaRESUMEN
In meiosis, crossovers between homologous chromosomes link them together. This enables them to attach to microtubules of the meiotic spindle as a unit, such that the homologs will be pulled away from one another at anaphase I. Homologous pairs can sometimes fail to become linked by crossovers. In some organisms, these non-exchange partners are still able to segregate properly. In several organisms, associations between the centromeres of non-exchange partners occur in meiotic prophase. These associations have been proposed to promote segregation in meiosis I. But it is unclear how centromere pairing could promote subsequent proper segregation. Here we report that meiotic centromere pairing of chromosomes in mouse spermatocytes allows the formation of an association between chromosome pairs. We find that heterochromatin regions of homologous centromeres remain associated even after centromere-pairing dissolves. Our results suggest the model that, in mouse spermatocytes, heterochromatin maintains the association of homologous centromeres in the absence crossing-over.
Asunto(s)
Centrómero , Emparejamiento Cromosómico , Segregación Cromosómica , Heterocromatina , Meiosis , Espermatocitos , Animales , Masculino , Ratones , Profase , Recombinación GenéticaRESUMEN
Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.
Asunto(s)
Anafase/fisiología , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Segregación Cromosómica/fisiología , Profase/fisiología , Espermatocitos/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Centrómero/genética , Masculino , Ratones , Ratones Noqueados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espermatocitos/citología , Huso Acromático/genética , Huso Acromático/metabolismo , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure.
Asunto(s)
Intercambio Genético , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Meiosis/genética , Animales , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Recombinación Genética , Espermatocitos/metabolismoRESUMEN
The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación/fisiología , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Factores de Transcripción/genéticaRESUMEN
During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Emparejamiento Cromosómico , Cromosomas/química , Meiosis , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Cromosomas/ultraestructura , Ratones , Mitosis , Complejo Sinaptonémico , CohesinasRESUMEN
Meiosis produces haploid gametes through a succession of chromosomal events, including pairing, synapsis, and recombination. Mechanisms that orchestrate these events remain poorly understood. We found that the SUMO (small ubiquitin-like modifier)-modification and ubiquitin-proteasome systems regulate the major events of meiotic prophase in mouse. Interdependent localization of SUMO, ubiquitin, and proteasomes along chromosome axes was mediated largely by RNF212 and HEI10, two E3 ligases that are also essential for crossover recombination. RNF212-dependent SUMO conjugation effected a checkpointlike process that stalls recombination by rendering the turnover of a subset of recombination factors dependent on HEI10-mediated ubiquitylation. We propose that SUMO conjugation establishes a precondition for designating crossover sites via selective protein stabilization. Thus, meiotic chromosome axes are hubs for regulated proteolysis via SUMO-dependent control of the ubiquitin-proteasome system.
Asunto(s)
Intercambio Genético/fisiología , Ligasas/metabolismo , Meiosis/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de Ciclo Celular , Emparejamiento Cromosómico , Cromosomas de los Mamíferos/metabolismo , Intercambio Genético/genética , Ligasas/genética , Masculino , Meiosis/genética , Ratones , Ratones Mutantes , Proteolisis , Espermatocitos/citología , Espermatocitos/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.
Asunto(s)
Disparidad de Par Base , Proteínas de Ciclo Celular/metabolismo , Reparación de la Incompatibilidad de ADN , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Transactivadores/metabolismoRESUMEN
During prophase of meiosis I, homologous chromosomes interact and undergo recombination. Successful completion of these processes is required in order for the homologous chromosomes to mount the meiotic spindle as a pair. The organization of the chromosomes into pairs ensures orderly segregation to opposite poles of the dividing cell, such that each gamete receives one copy of each chromosome. Chiasmata, the cytological manifestation of crossover products of recombination, physically connect the homologs in pairs, providing a linkage that facilitates their segregation. Consequently, mutations that reduce the level of recombination are invariably associated with increased errors in meiotic chromosome segregation. In this review, we focus on recent biochemical and genetic advances in elucidating the mechanisms of meiotic DNA strand exchange catalyzed by the Dmc1 protein. We also discuss the mode by which two recombination mediators, Hop2 and Mnd1, facilitate rate-limiting steps of DNA strand exchange catalyzed by Dmc1.
Asunto(s)
Reparación del ADN , Meiosis , Recombinación Genética , Animales , Proteínas de Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/fisiología , Humanos , Proteínas Nucleares/fisiología , Recombinasa Rad51/fisiología , Transactivadores/fisiologíaRESUMEN
Telomere-led rapid prophase movements (RPMs) in meiotic prophase have been observed in diverse eukaryote species. A shared feature of RPMs is that the force that drives the chromosomal movements is transmitted from the cytoskeleton, through the nuclear envelope, to the telomeres. Studies in mice suggested that dynein movement along microtubules is transmitted to telomeres through SUN1/KASH5 nuclear envelope bridges to generate RPMs. We monitored RPMs in mouse seminiferous tubules using 4D fluorescence imaging and quantitative motion analysis to characterize patterns of movement in the RPM process. We find that RPMs reflect a combination of nuclear rotation and individual chromosome movements. The telomeres move along microtubule tracks that are apparently continuous with the cytoskeletal network and exhibit characteristic arrangements at different stages of prophase. Quantitative measurements confirmed that SUN1/KASH5, microtubules, and dynein, but not actin, were necessary for RPMs and that defects in meiotic recombination and synapsis resulted in altered RPMs.
Asunto(s)
Profase , Túbulos Seminíferos/ultraestructura , Telómero/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Proteínas Nucleares/metabolismo , Túbulos Seminíferos/metabolismo , Telómero/ultraestructuraRESUMEN
The HOP2-MND1 heterodimer is required for progression of homologous recombination in eukaryotes. In vitro, HOP2-MND1 stimulates the DNA strand exchange activities of RAD51 and DMC1. We demonstrate that HOP2-MND1 induces changes in the conformation of RAD51 that profoundly alter the basic properties of RAD51. HOP2-MND1 enhances the interaction of RAD51 with nucleotide cofactors and modifies its DNA-binding specificity in a manner that stimulates DNA strand exchange. It enables RAD51 DNA strand exchange in the absence of divalent metal ions required for ATP binding and offsets the effect of the K133A mutation that disrupts ATP binding. During nucleoprotein formation HOP2-MND1 helps to load RAD51 on ssDNA restricting its dsDNA-binding and during the homology search it promotes dsDNA binding removing the inhibitory effect of ssDNA. The magnitude of the changes induced in RAD51 defines HOP2-MND1 as a 'molecular trigger' of RAD51 DNA strand exchange.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Nucleótidos/metabolismo , Recombinasa Rad51/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Ciclo Celular/genética , ADN/genética , Ratones , Unión Proteica , Recombinasa Rad51/genéticaRESUMEN
The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , ADN/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Sitios de Unión/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Homología de Secuencia de Aminoácido , Soluciones/químicaRESUMEN
Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2-MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1(-/-) spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2-MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Meiosis/genética , Recombinasas/metabolismo , Reparación del ADN por Recombinación , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular , Emparejamiento Cromosómico , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Proteínas de Unión a FosfatoRESUMEN
The Hop2-Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2-Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2-Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2-Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Recombinación Homóloga , Meiosis/genética , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Mutación Puntual , Multimerización de Proteína , Estructura Terciaria de Proteína , Recombinasas/metabolismoRESUMEN
Observations of a wide range of organisms show that the centromeres form associations of pairs or small groups at different stages of meiotic prophase. Little is known about the functions or mechanisms of these associations, but in many cases, synaptonemal complex elements seem to play a fundamental role. Two main associations are observed: homology-independent associations very early in the meiotic program-sometimes referred to as centromere coupling-and a later association of homologous centromeres, referred to as centromere pairing or tethering. The later centromere pairing initiates during synaptonemal complex assembly, then persists after the dissolution of the synaptonemal complex. While the function of the homology-independent centromere coupling remains a mystery, centromere pairing appears to have a direct impact on the chromosome segregation fidelity of achiasmatic chromosomes. Recent work in yeast, Drosophila, and mice suggest that centromere pairing is a previously unappreciated, general meiotic feature that may promote meiotic segregation fidelity of the exchange and non-exchange chromosomes.