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Scientific Reports Apr 2024DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication...
DNA double-strand breaks (DSBs) activate DNA damage responses (DDRs) in both mitotic and meiotic cells. A single-stranded DNA (ssDNA) binding protein, Replication protein-A (RPA) binds to the ssDNA formed at DSBs to activate ATR/Mec1 kinase for the response. Meiotic DSBs induce homologous recombination monitored by a meiotic DDR called the recombination checkpoint that blocks the pachytene exit in meiotic prophase I. In this study, we further characterized the essential role of RPA in the maintenance of the recombination checkpoint during Saccharomyces cerevisiae meiosis. The depletion of an RPA subunit, Rfa1, in a recombination-defective dmc1 mutant, fully alleviates the pachytene arrest with the persistent unrepaired DSBs. RPA depletion decreases the activity of a meiosis-specific CHK2 homolog, Mek1 kinase, which in turn activates the Ndt80 transcriptional regulator for pachytene exit. These support the idea that RPA is a sensor of ssDNAs for the activation of meiotic DDR. Rfa1 depletion also accelerates the prophase I delay in the zip1 mutant defective in both chromosome synapsis and the recombination, consistent with the notion that the accumulation of ssDNAs rather than defective synapsis triggers prophase I delay in the zip1 mutant.
Topics: Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Meiosis; DNA Breaks, Double-Stranded; Cell Cycle Proteins; DNA-Binding Proteins; Recombination, Genetic; Homologous Recombination; MAP Kinase Kinase 1; DNA, Single-Stranded; Nuclear Proteins; Transcription Factors
PubMed: 38664461
DOI: 10.1038/s41598-024-60082-x -
Plant & Cell Physiology Sep 2023Plant cells lack centrosomes and instead utilize acentrosomal microtubule organizing centers (MTOCs) to rapidly increase the number of microtubules at the onset of...
Plant cells lack centrosomes and instead utilize acentrosomal microtubule organizing centers (MTOCs) to rapidly increase the number of microtubules at the onset of spindle assembly. Although several proteins required for MTOC formation have been identified, how the MTOC is positioned at the right place is not known. Here, we show that the inner nuclear membrane protein SUN2 is required for MTOC association with the nuclear envelope (NE) during mitotic prophase in the moss Physcomitrium patens. In actively dividing protonemal cells, microtubules accumulate around the NE during prophase. In particular, regional MTOC is formed at the apical surface of the nucleus. However, microtubule accumulation around the NE was impaired and apical MTOCs were mislocalized in sun2 knockout cells. Upon NE breakdown, the mitotic spindle was assembled with mislocalized MTOCs. However, completion of chromosome alignment in the spindle was delayed; in severe cases, the chromosome was transiently detached from the spindle body. SUN2 tended to localize to the apical surface of the nucleus during prophase in a microtubule-dependent manner. Based on these results, we propose that SUN2 facilitates the attachment of microtubules to chromosomes during spindle assembly by localizing microtubules to the NE. MTOC mispositioning was also observed during the first division of the gametophore tissue. Thus, this study suggests that microtubule-nucleus linking, a well-known function of SUN in animals and yeast, is conserved in plants.
Topics: Animals; Nuclear Envelope; Microtubule-Organizing Center; Microtubules; Spindle Apparatus; Chromosomes; Bryopsida
PubMed: 37421143
DOI: 10.1093/pcp/pcad074 -
HGG Advances Jan 2024In this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified...
In this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified that the CEP192 biallelic variants (c.1912C>T, p.His638Tyr and c.5750A>G, p.Asn1917Ser) segregated with microcephaly, short stature, limb-extremity dysplasia, and reduced testicular size, while CEP192 monoallelic variants segregated with infertility and/or reduced testicular size in the family. In 1,264 unrelated patients, variant screening for CEP192 identified a same variant (c.5750A>G, p.Asn1917Ser) and other variants significantly associated with infertility. Two lines of Cep192 mice model that are equivalent to human variants were generated. Embryos with Cep192 biallelic variants arrested at E7 because of cell apoptosis mediated by MVA/tetraploidy cell acumination. Mice with heterozygous variants replicated the predisposition to male infertility. Mouse primary embryonic fibroblasts with Cep192 biallelic variants cultured in vitro showed abnormal morphology, mitotic arresting, and disruption of spindle formation. In patient epithelial cells with biallelic variants cultured in vitro, the number of cells arrested during the prophase increased because of the failure of spindle formation. Accordingly, we present mutant CEP192, which is a link for the MVA syndrome with tetraploidy and the predisposition to male infertility.
Topics: Humans; Male; Mice; Animals; Tetraploidy; Aneuploidy; Chromosome Disorders; Disease Susceptibility; Infertility, Male; Chromosomal Proteins, Non-Histone; Mosaicism
PubMed: 37981762
DOI: 10.1016/j.xhgg.2023.100256 -
Nucleic Acids Research Apr 2024Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate...
Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate 3' single-stranded DNA (ssDNA) tails, one DSB end engages a homolog partner chromatid and is extended by DNA synthesis, whereas the other end remains associated with its sister. Then, after regulated differentiation into crossover- and noncrossover-fated types, the second DSB end participates in the reaction by strand annealing with the extended first end, along both pathways. This second-end capture is dependent on Rad52, presumably via its known capacity to anneal two ssDNAs. Here, using physical analysis of DNA recombination, we demonstrate that this process is dependent on direct interaction of Rad52 with the ssDNA binding protein, replication protein A (RPA). Furthermore, the absence of this Rad52-RPA joint activity results in a cytologically-prominent RPA spike, which emerges from the homolog axes at sites of crossovers during the pachytene stage of the meiotic prophase. Our findings suggest that this spike represents the DSB end of a broken chromatid caused by either the displaced leading DSB end or the second DSB end, which has been unable to engage with the partner homolog-associated ssDNA. These and other results imply a close correspondence between Rad52-RPA roles in meiotic recombination and mitotic DSB repair.
Topics: Rad52 DNA Repair and Recombination Protein; Replication Protein A; Meiosis; Saccharomyces cerevisiae Proteins; Crossing Over, Genetic; Saccharomyces cerevisiae; DNA Breaks, Double-Stranded; Recombination, Genetic; DNA, Single-Stranded; Homologous Recombination
PubMed: 38340339
DOI: 10.1093/nar/gkae083 -
The Journal of Biological Chemistry Jun 2024Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in...
Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.
PubMed: 38844130
DOI: 10.1016/j.jbc.2024.107446