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Molecular Biology of the Cell May 2022Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation....
Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1-4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated in early prophase spermatocytes and assessed stages of HR, crossover formation, and SC processes. mutation resulted in increased RPA foci and reduced RAD51/DMC1 foci during zygonema, and an increase of both class I and class II crossover events. Furthermore, the disassembly of SC lateral elements was aberrant. Our results highlight the importance of PLK1 in regulating HR and SC disassembly during spermatogenesis.
Topics: Animals; Cell Cycle Proteins; Chromosome Pairing; DNA; Homologous Recombination; Male; Mammals; Meiosis; Mice; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Spermatogenesis; Synaptonemal Complex; Polo-Like Kinase 1
PubMed: 35274968
DOI: 10.1091/mbc.E21-03-0115 -
Molecular Cell Apr 2024Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining...
Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.
Topics: Animals; Chromatin; Nuclear Proteins; Transcription Factors; DNA; DNA End-Joining Repair; Histones; Chromosome Pairing; Ku Autoantigen; Mammals
PubMed: 38423014
DOI: 10.1016/j.molcel.2024.02.002 -
International Journal of Molecular... Dec 2021The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA...
The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.
Topics: Binding Sites; Chromosome Pairing; DNA; DNA Restriction Enzymes; Plasmids; Protein Binding; Proteins; Synaptosomes
PubMed: 35008637
DOI: 10.3390/ijms23010212 -
American Journal of Human Genetics Feb 2021Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still...
Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.
Topics: Adult; Azoospermia; Chromosome Pairing; Codon, Nonsense; DNA-Binding Proteins; Female; Homozygote; Humans; Male; Meiosis; Middle Aged; Mutation; Nuclear Proteins; Pedigree; Primary Ovarian Insufficiency; Spermatocytes; Synaptonemal Complex; Whole Genome Sequencing
PubMed: 33508233
DOI: 10.1016/j.ajhg.2021.01.010 -
Science Advances Oct 2023In almost all sexually reproducing organisms, meiotic recombination and cell division require the synapsis of homologous chromosomes by a large proteinaceous structure,...
In almost all sexually reproducing organisms, meiotic recombination and cell division require the synapsis of homologous chromosomes by a large proteinaceous structure, the synaptonemal complex (SC). While the SC's overall structure is highly conserved across eukaryotes, its constituent proteins diverge between phyla. Transverse filament protein, SYCP1, spans the width of the SC and undergoes amino-terminal head-to-head self-assembly in vitro through a motif that is unusually highly conserved across kingdoms of life. Here, we report creation of mouse mutants, and , that target SYCP1's head-to-head interface. L106E resulted in a complete loss of synapsis, while L102E had no apparent effect on synapsis, in agreement with their differential effects on the SYCP1 head-to-head interface in molecular dynamics simulations. In mice, homologs aligned and recruited low levels of mutant SYCP1 and other SC proteins, but the absence of synapsis led to failure of crossover formation and meiotic arrest. We conclude that SYCP1's conserved head-to-head interface is essential for meiotic chromosome synapsis in vivo.
Topics: Animals; Mice; Chromosome Pairing; Homologous Recombination; Meiosis; Nuclear Proteins; Synaptonemal Complex
PubMed: 37862414
DOI: 10.1126/sciadv.adi1562 -
International Journal of Molecular... Aug 2022Meiosis initiates with the formation of double strand breaks (DSBs) throughout the genome. To avoid genomic instability, these DSBs need to be correctly repaired by...
Meiosis initiates with the formation of double strand breaks (DSBs) throughout the genome. To avoid genomic instability, these DSBs need to be correctly repaired by homologous recombination. Surveillance mechanisms involving the DNA damage response (DDR) pathway ATM-CHK2-p53 can detect the persistence of unrepaired DBSs and activate the recombination-dependent arrest at the pachytene stage. However, a complete understanding of p53 functions under normal physiological conditions remains lacking. Here, we report a detailed analysis of the p53 role during meiotic prophase in mice spermatocytes. We show that the absence of p53 regulates prophase progression by slowing down the pachytene stage when the recombination-dependent arrest occurs. Furthermore, our results show that p53 is necessary for proper crossover (CO) formation and localization. Our study contributes to a deeper understanding of p53 roles during the meiotic prophase.
Topics: Animals; Cell Cycle Proteins; DNA Breaks, Double-Stranded; Male; Meiosis; Mice; Prophase; Spermatocytes; Tumor Suppressor Protein p53
PubMed: 36077210
DOI: 10.3390/ijms23179818 -
The Journal of Steroid Biochemistry and... Oct 2022Testosterone (T) and dihydrotestosterone (DHT) are the main hormones regulating reproduction and development of male animals. Although their synthesis and secretion are...
Testosterone (T) and dihydrotestosterone (DHT) are the main hormones regulating reproduction and development of male animals. Although their synthesis and secretion are regulated by the endocrine system [hypothalamic-pituitary-gonadal (adrenal) axis], it is also possible to synthesize T and DHT from the induction of two proteins: Syce1 and Syce3. As central elements of the synaptonemal complex (SC), Syce1 and Syce3 play a key role in the association of homologous chromosomes during meiosis. However, Syce1 and Syce3 also promote the synthesis of T and DHT, although potential mechanisms have yet to be revealed. In this study, Leydig and Sertoli cells, which are responsible for the production and regulation of steroid hormones in testis, were transfected with recombinant Syce1/Syce3 and silence sequence. Our results revealed the highest expression of Syce1 and Syce3 in spermatogenic cells of the testis. Moreover, overexpression or knockdown of Syce1 and Syce3 in Sertoli and Leydig cells resulted in activation or suppression of steroidogenic genes Star and Hsd3b, which are involved in a steroidogenic pathway that upregulates T synthesis. Upregulated expression of Syce1 resulted in a significant increase in Srd5a1, which can promote DHT secretion. Interestingly, Syce1 and Syce3 overexpression synergistically promoted each other's abundance. Our results define a previously unknown mechanism of Syce1 and Syce3 dependent activation of steroidogenic signaling in Sertoli and Leydig cells.
Topics: Animals; Dihydrotestosterone; Leydig Cells; Male; Mice; Sertoli Cells; Synaptonemal Complex; Testis; Testosterone; Testosterone Congeners
PubMed: 35697131
DOI: 10.1016/j.jsbmb.2022.106135 -
Current Genetics Oct 2020Pairing of homologous chromosomes is crucial for ensuring accurate segregation of chromosomes during meiosis. Molecular mechanisms of homologous chromosome pairing in... (Review)
Review
Pairing of homologous chromosomes is crucial for ensuring accurate segregation of chromosomes during meiosis. Molecular mechanisms of homologous chromosome pairing in meiosis have been extensively studied in the fission yeast Schizosaccharomyces pombe. In this organism, meiosis-specific noncoding RNA transcribed from specific genes accumulates at the respective gene loci, and chromosome-associated RNA-protein complexes mediate meiotic pairing of homologous loci through phase separation. Pairing of homologous chromosomes also occurs in somatic diploid cells in certain situations. For example, somatic pairing of homologous chromosomes occurs during the early embryogenesis in diptera, and relies on the transcription-associated chromatin architecture. Earlier models also suggest that transcription factories along the chromosome mediate pairing of homologous chromosomes in plants. These studies suggest that RNA bodies formed on chromosomes mediate the pairing of homologous chromosomes. This review summarizes lessons from S. pombe to provide general insights into mechanisms of homologous chromosome pairing mediated by phase separation of chromosome-associated RNA-protein complexes.
Topics: Animals; Chromosome Pairing; Chromosomes; Chromosomes, Fungal; DNA; Drosophila melanogaster; Meiosis; RNA, Fungal; RNA, Untranslated; RNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Sequence Homology, Nucleic Acid
PubMed: 32285141
DOI: 10.1007/s00294-020-01077-9 -
Frontiers in Endocrinology 2023The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources....
INTRODUCTION
The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin.
METHODS
To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific knockout ( cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed.
RESULTS
The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes.
DISCUSSION
These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.
Topics: Animals; Female; Mice; Cisplatin; Cyclophosphamide; DNA; Oocytes; Ovarian Follicle; Prophase; Ku Autoantigen
PubMed: 37900135
DOI: 10.3389/fendo.2023.1268009 -
Nucleus (Austin, Tex.) Dec 2019Active meiotic chromosome movements are a universally conserved feature. They occur at the early stages of prophase of the first meiotic division and support the... (Review)
Review
Active meiotic chromosome movements are a universally conserved feature. They occur at the early stages of prophase of the first meiotic division and support the chromosome pairing process by (1) efficiently installing the synaptonemal complex between homologous chromosomes, (2) discouraging inadvertent chromosome interactions and (3) bringing homologous chromosomes into proximity. Chromosome movements are driven by forces in the cytoplasm, which are passed on to chromosome ends attached to the nuclear periphery by nuclear-membrane-spanning protein modules. In this extra view, we highlight our recent studies into the role of the nuclear lamina during this process to emphasize that it is a highly conserved structure in metazoans. The nuclear lamina forms a rigid proteinaceous network that underlies the inner nuclear membrane to provide stability to the nucleus. Misdemeanors of the nuclear lamina during meiosis has deleterious consequences for the viability and health of the offspring, highlighting the importance of a functional nuclear lamina during this cell cycle stage. Abbreviations: DSB: DNA double strand break; LEM: LAP2, Emerin, MAN1; LINC: LInker of the Nucleoskeleton and Cytoskeleton; RPM: rapid prophase movement; SUN/KASH: Sad1p, UNC-84/Klarsicht, ANC-1, Syne Homology.
Topics: Animals; Chromosomes; Humans; Lamins; Meiosis
PubMed: 30676220
DOI: 10.1080/19491034.2019.1572413