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1.
Cell Mol Life Sci ; 81(1): 194, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38653846

ABSTRACT

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.


Subject(s)
Chromosome Pairing , Chromosome Segregation , Meiosis , Humans , Animals , Chromosome Pairing/genetics , Male , Meiosis/genetics , Mice , Chromosome Segregation/genetics , Female , Aneuploidy , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/genetics , Sex Chromosomes/genetics , Crossing Over, Genetic/genetics
2.
bioRxiv ; 2024 May 30.
Article in English | MEDLINE | ID: mdl-38463951

ABSTRACT

Double-strand breaks (DSBs) are the most deleterious lesions experienced by our genome. Yet, DSBs are intentionally induced during gamete formation to promote the exchange of genetic material between homologous chromosomes. While the conserved topoisomerase-like enzyme Spo11 catalyzes DSBs, additional regulatory proteins-referred to as "Spo11 accessory factors"- regulate the number, timing, and placement of DSBs during early meiotic prophase ensuring that SPO11 does not wreak havoc on the genome. Despite the importance of the accessory factors, they are poorly conserved at the sequence level suggesting that these factors may adopt unique functions in different species. In this work, we present a detailed analysis of the genetic and physical interactions between the DSB factors in the nematode Caenorhabditis elegans providing new insights into conserved and novel functions of these proteins. This work shows that HIM-5 is the determinant of X-chromosome-specific crossovers and that its retention in the nucleus is dependent on DSB-1, the sole accessory factor that interacts with SPO-11. We further provide evidence that HIM-5 coordinates the actions of the different accessory factors sub-groups, providing insights into how components on the DNA loops may interact with the chromosome axis.

3.
Proc Natl Acad Sci U S A ; 120(47): e2310951120, 2023 Nov 21.
Article in English | MEDLINE | ID: mdl-37976262

ABSTRACT

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine whether and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114 and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.


Subject(s)
Chromosomes , Homologous Recombination , Animals , Male , Mice , Homologous Recombination/genetics , Meiosis/genetics , Mutation , Spermatogenesis/genetics
4.
Cell Mol Life Sci ; 80(10): 279, 2023 Sep 08.
Article in English | MEDLINE | ID: mdl-37682311

ABSTRACT

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11ß splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11ßki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11ßki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11ß and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.


Subject(s)
Endodeoxyribonucleases , Pseudoautosomal Regions , Animals , Humans , Male , Mice , Alleles , Protein Isoforms/genetics , Recombination, Genetic/genetics , Semen , Endodeoxyribonucleases/genetics
5.
bioRxiv ; 2023 Jul 20.
Article in English | MEDLINE | ID: mdl-37162821

ABSTRACT

Meiotic DNA double-strand breaks (DSBs) initiate homologous recombination and are crucial for ensuring proper chromosome segregation. In mice, ANKRD31 recently emerged as a regulator of DSB timing, number, and location, with a particularly important role in targeting DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. ANKRD31 interacts with multiple proteins, including the conserved and essential DSB-promoting factor REC114, so it was hypothesized to be a modular scaffold that "anchors" other proteins together and to meiotic chromosomes. To determine if and why the REC114 interaction is important for ANKRD31 function, we generated mice with Ankrd31 mutations that either reduced (missense mutation) or eliminated (C-terminal truncation) the ANKRD31-REC114 interaction without diminishing contacts with other known partners. A complete lack of the ANKRD31-REC114 interaction mimicked an Ankrd31 null, with delayed DSB formation and recombination, defects in DSB repair, and altered DSB locations including failure to target DSBs to the PARs. In contrast, when the ANKRD31-REC114 interaction was substantially but not completely disrupted, spermatocytes again showed delayed DSB formation globally, but recombination and repair were hardly affected and DSB locations were similar to control mice. The missense Ankrd31 allele showed a dosage effect, wherein combining it with the null or C-terminal truncation allele resulted in intermediate phenotypes for DSB formation, recombination, and DSB locations. Our results show that ANKRD31 function is critically dependent on its interaction with REC114, and that defects in ANKRD31 activity correlate with the severity of the disruption of the interaction.

6.
Cell Mol Life Sci ; 80(4): 107, 2023 Mar 26.
Article in English | MEDLINE | ID: mdl-36967403

ABSTRACT

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.


Subject(s)
Lysine , RNA-Binding Protein FUS , Animals , Male , Lysine/genetics , RNA-Binding Protein FUS/genetics , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Homologous Recombination , DNA/metabolism , Meiosis/genetics , Mammals/metabolism
7.
Plant Reprod ; 36(1): 73-95, 2023 03.
Article in English | MEDLINE | ID: mdl-36646915

ABSTRACT

Meiosis is a highly conserved specialised cell division in sexual life cycles of eukaryotes, forming the base of gene reshuffling, biological diversity and evolution. Understanding meiotic machinery across different plant lineages is inevitable to understand the lineage-specific evolution of meiosis. Functional and cytogenetic studies of meiotic proteins from all plant lineage representatives are nearly impossible. So, we took advantage of the genomics revolution to search for core meiotic proteins in accumulating plant genomes by the highly sensitive homology search approaches, PSI-BLAST, HMMER and CLANS. We could find that most of the meiotic proteins are conserved in most of the lineages. Exceptionally, Arabidopsis thaliana ASY4, PHS1, PRD2, PRD3 orthologs were mostly not detected in some distant algal lineages suggesting their minimal conservation. Remarkably, an ancestral duplication of SPO11 to all eukaryotes could be confirmed. Loss of SPO11-1 in Chlorophyta and Charophyta is likely to have occurred, suggesting that SPO11-1 and SPO11-2 heterodimerisation may be a unique feature in land plants of Viridiplantae. The possible origin of the meiotic proteins described only in plants till now, DFO and HEIP1, could be traced and seems to occur in the ancestor of vascular plants and Streptophyta, respectively. Our comprehensive approach is an attempt to provide insights about meiotic core proteins and thus the conservation of meiotic pathways across plant kingdom. We hope that this will serve the meiotic community a basis for further characterisation of interesting candidates in future.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Genome, Plant , Meiosis , Cell Cycle Proteins/genetics
8.
Curr Top Dev Biol ; 151: 27-42, 2023.
Article in English | MEDLINE | ID: mdl-36681473

ABSTRACT

Recent discoveries have advanced our understanding of recombination initiation beyond the placement of double-stranded DNA breaks (DSBs) from germline replication timing to the dynamic reorganization of chromatin, and defined critical players of recombination initiation. This article focuses on recombination initiation in mammals utilizing the PRDM9 protein to orchestrate crucial stages of meiotic recombination initiation by interacting with the local DNA environment and several protein complexes. The Pioneer Complex with the SNF2-type chromatin remodeling enzyme HELLS, exposes PRDM9-bound DNA. At the same time, a Compass-Complex containing EWSR1, CXXC1, CDYL, EHMT2 and PRDM9 facilitates the association of putative hotspot sites in DNA loops with the chromosomal axis where DSB-promoting complexes are located, and DSBs are catalyzed by the SPO11/TOPOVIBL complex. Finally, homology search is facilitated at PRDM9-directed sites by ANKRD31. The Reader-Writer system consists of PRDM9 writing characteristic histone methylation signatures, which are read by ZCWPW1, promoting efficient homology engagement.


Subject(s)
Chromatin , DNA , Animals , DNA/metabolism , Chromosomes , Homologous Recombination , DNA Breaks, Double-Stranded , Meiosis/genetics , Mammals/genetics
9.
Plant Biotechnol J ; 21(2): 405-418, 2023 02.
Article in English | MEDLINE | ID: mdl-36373224

ABSTRACT

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.


Subject(s)
CRISPR-Cas Systems , Triticum , Triticum/genetics , CRISPR-Cas Systems/genetics , Plant Breeding , Chromosomes , Meiosis/genetics
10.
Genes Cells ; 28(2): 129-148, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36530025

ABSTRACT

Deficiency in meiotic recombination leads to aberrant chromosome disjunction during meiosis, often resulting in the lethality of gametes or genetic disorders due to aneuploidy formation. Budding yeasts lacking Spo11, which is essential for initiation of meiotic recombination, produce many inviable spores in meiosis, while very rarely all sets of 16 chromosomes are coincidentally assorted into gametes to form viable spores. We induced meiosis in a spo11∆ diploid, in which homolog pairs can be distinguished by single nucleotide polymorphisms and determined whole-genome sequences of their exceptionally viable spores. We detected no homologous recombination in the viable spores of spo11∆ diploid. Point mutations were fewer in spo11∆ than in wild-type. We observed spo11∆ viable spores carrying a complete diploid set of homolog pairs or haploid spores with a complete haploid set of homologs but with aneuploidy in some chromosomes. In the latter, we found the chromosome-dependence in the aneuploid incidence, which was positively and negatively influenced by the chromosome length and the impact of dosage-sensitive genes, respectively. Selection of aneuploidy during meiosis II or mitosis after spore germination was also chromosome dependent. These results suggest a pathway by which specific chromosomes are more prone to cause aneuploidy, as observed in Down syndrome.


Subject(s)
Aneuploidy , Meiosis , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Chromosomes/metabolism , Endodeoxyribonucleases/genetics , Homologous Recombination , Meiosis/genetics , Meiosis/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
11.
J Mol Cell Biol ; 14(11)2023 04 06.
Article in English | MEDLINE | ID: mdl-36484653

ABSTRACT

Spermatogenesis is a highly complex developmental process that typically consists of mitosis, meiosis, and spermiogenesis. DNA/RNA helicase DHX36, a unique guanine-quadruplex (G4) resolvase, plays crucial roles in a variety of biological processes. We previously showed that DHX36 is highly expressed in male germ cells with the highest level in zygotene spermatocytes. Here, we deleted Dhx36 in advanced germ cells with Stra8-GFPCre and found that a Dhx36 deficiency in the differentiated spermatogonia leads to meiotic defects and abnormal spermiogenesis. These defects in late stages of spermatogenesis arise from dysregulated transcription of G4-harboring genes, which are required for meiosis. Thus, this study reveals that Dhx36 plays crucial roles in late stages of spermatogenesis.


Subject(s)
RNA Helicases , RNA , Male , DNA/genetics , DNA Helicases/genetics , Meiosis , RNA Helicases/genetics , Spermatocytes , Spermatogenesis/genetics , Animals , Mice
12.
Biomolecules ; 12(12)2022 11 26.
Article in English | MEDLINE | ID: mdl-36551189

ABSTRACT

Meiotic recombination is a pivotal event that ensures faithful chromosome segregation and creates genetic diversity in gametes. Meiotic recombination is initiated by programmed double-strand breaks (DSBs), which are catalyzed by the conserved Spo11 protein. Spo11 is an enzyme with structural similarity to topoisomerase II and induces DSBs through the nucleophilic attack of the phosphodiester bond by the hydroxy group of its tyrosine (Tyr) catalytic residue. DSBs caused by Spo11 are repaired by homologous recombination using homologous chromosomes as donors, resulting in crossovers/chiasmata, which ensure physical contact between homologous chromosomes. Thus, the site of meiotic recombination is determined by the site of the induced DSB on the chromosome. Meiotic recombination is not uniformly induced, and sites showing high recombination rates are referred to as recombination hotspots. In fission yeast, ade6-M26, a nonsense point mutation of ade6 is a well-characterized meiotic recombination hotspot caused by the heptanucleotide sequence 5'-ATGACGT-3' at the M26 mutation point. In this review, we summarize the meiotic recombination mechanisms revealed by the analysis of the fission ade6-M26 gene as a model system.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Homologous Recombination , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Models, Biological
13.
Mol Biol Evol ; 39(11)2022 11 03.
Article in English | MEDLINE | ID: mdl-36256608

ABSTRACT

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.


Subject(s)
Archaeal Proteins , DNA Topoisomerases, Type II , Phylogeny , Amino Acid Sequence , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Archaeal Proteins/chemistry , Meiosis/genetics , Eukaryota/genetics , Eukaryota/metabolism
14.
Chromosome Res ; 30(4): 415-428, 2022 12.
Article in English | MEDLINE | ID: mdl-35674907

ABSTRACT

Most plant species have three or more SPO11/TOPOVIA homologs and two TOPOVIB homologs, which associate to trigger meiotic double-strand break (DSB) formation and subsequent meiotic recombination. In Zea mays L. (maize), ZmSPO11-1 and ZmMTOPVIB have been reported to be indispensable for the initiation of meiotic recombination, yet the function of ZmSPO11-2 remains unclear. In this study, we characterized meiotic functions of ZmSPO11-2 during male meiosis in maize. Two independent Zmspo11-1 knock-out mutants exhibited normal vegetative growth but both male and female sterility. The formation of meiotic DSBs of DNA molecules was fully abolished in the Zmspo11-2 plants, leading to the defective homologous chromosome paring, synapsis, recombination, and segregation. However, the bipolar spindle assembly was not noticeably affected in Zmspo11-2 meiocytes. Overall, our results demonstrate that as its partner ZmSPO11-1 and ZmMTOPVIB, ZmSPO11-2 plays essential roles in DSB formation and homologous recombination in maize meiosis.


Subject(s)
DNA Breaks, Double-Stranded , Zea mays , Zea mays/genetics , Meiosis , Homologous Recombination , Chromosomes
15.
Biochim Biophys Acta Mol Cell Res ; 1869(6): 119239, 2022 06.
Article in English | MEDLINE | ID: mdl-35181406

ABSTRACT

Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity.


Subject(s)
Paramecium tetraurelia , Germ Cells , Macronucleus/genetics , Macronucleus/metabolism , Meiosis/genetics , Paramecium tetraurelia/genetics , Paramecium tetraurelia/metabolism , RNA, Untranslated/metabolism
16.
Int J Mol Sci ; 22(21)2021 Nov 02.
Article in English | MEDLINE | ID: mdl-34769330

ABSTRACT

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.


Subject(s)
Cell Differentiation , Cysts/pathology , Endodeoxyribonucleases/metabolism , Gene Expression Regulation , Protozoan Proteins/metabolism , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cysts/genetics , Cysts/metabolism , Endodeoxyribonucleases/genetics , Giardia lamblia , Promoter Regions, Genetic , Protozoan Proteins/genetics
17.
Cell ; 184(24): 5970-5984.e18, 2021 11 24.
Article in English | MEDLINE | ID: mdl-34793701

ABSTRACT

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.


Subject(s)
Gene Deletion , Gene Duplication , Germ Cells/metabolism , Recombination, Genetic/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/deficiency , Ataxia Telangiectasia Mutated Proteins/metabolism , Base Sequence , Chromatids/metabolism , Chromosomes, Mammalian/genetics , Crosses, Genetic , DNA Breaks, Double-Stranded , DNA, Circular/genetics , Female , Genome , Haplotypes/genetics , Homologous Recombination/genetics , Male , Mice, Inbred C57BL , Mice, Inbred DBA , Mutagenesis, Insertional/genetics , Mutation/genetics
18.
Int J Mol Sci ; 22(17)2021 Aug 28.
Article in English | MEDLINE | ID: mdl-34502253

ABSTRACT

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic SPO11 are indispensable in a functional form for double strand break induction during meiotic prophase I. This stands in contrast to mammals and fungi, where a single SPO11 is present and sufficient. We aimed to investigate the specific function and evolution of both meiotic SPO11 paralogs in land plants. By performing immunostaining of both SPO11-1 and -2, an investigation of the spatiotemporal localization of each SPO11 during meiosis was achieved. We further exchanged SPO11-1 and -2 in Arabidopsis and could show a species-specific function of the respective SPO11. By additional changes of regions between SPO11-1 and -2, a sequence-specific function for both the SPO11 proteins was revealed. Furthermore, the previous findings about the aberrant splicing of each SPO11 were refined by narrowing them down to a specific developmental phase. These findings let us suggest that the function of both SPO11 paralogs is highly sequence specific and that the orthologs are species specific.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Endodeoxyribonucleases/genetics , RNA Splicing/genetics , Arabidopsis/chemistry , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/physiology , Meiosis/physiology , Rad51 Recombinase/metabolism , Recombination, Genetic , Species Specificity
19.
Front Cell Dev Biol ; 9: 642737, 2021.
Article in English | MEDLINE | ID: mdl-33748134

ABSTRACT

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

20.
Chromosoma ; 130(1): 41-52, 2021 03.
Article in English | MEDLINE | ID: mdl-33492414

ABSTRACT

In mammalian oocytes, proper chromosome segregation at the first meiotic division is dictated by the presence and site of homologous chromosome recombination, which takes place in fetal life. Our current understanding of how homologous chromosomes find each other and initiate synapsis, which is prerequisite for homologous recombination, is limited. It is known that chromosome telomeres are anchored into the nuclear envelope (NE) at the early meiotic prophase I (MPI) and move along NE to facilitate homologous chromosome search and pairing. However, the mouse (Mus musculus) carries all acrocentric chromosomes with one telomeric end close to the centromere (subcentromeric telomere; C-telomere) and the other far away from the centromere (distal telomere; D-telomere), and how C- and D-telomeres participate in chromosome pairing and synapsis during the MPI progression is not well understood. Here, we found in the mouse oocyte that C- and D-telomeres transiently clustered in one area, but D-telomeres soon separated together from C-telomeres and then dispersed to preferentially initiate synapsis, while C-telomeres remained in clusters and synapsed at the last. In the Spo11 null oocyte, which is deficient in SPO11-dependent DSBs formation and homologous synapsis, the pattern of C- and D-telomere clustering and resolution was not affected, but synapsis was more frequently initiated at C-telomeres. These results suggest that SPO11 suppresses the early synapsis between C-telomeres in clusters.


Subject(s)
Chromosome Pairing , Chromosome Segregation , Chromosomes/genetics , Homologous Recombination , Meiotic Prophase I , Oocytes/physiology , Telomere , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centromere , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oocytes/cytology
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