Conservation and divergence of meiotic DNA double strand break forming mechanisms in Arabidopsis thaliana.
TL;DR: In this article, the authors show that MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, while PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between DSB-forming and resection machineries.
Abstract: In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.
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TL;DR: Crossovers also form a physical link between homologous chromosomes at metaphase I which is critical for accurate chromosome segregation and fertility as mentioned in this paper , and their regulation forms the focus of our current understanding of its regulation.
Abstract: Chromatin state, and dynamic loading of pro-crossover protein HEI10 at recombination intermediates shape meiotic chromosome patterning in plants. Meiosis is the basis of sexual reproduction, and its basic progression is conserved across eukaryote kingdoms. A key feature of meiosis is the formation of crossovers which result in the reciprocal exchange of segments of maternal and paternal chromosomes. This exchange generates chromosomes with new combinations of alleles, increasing the efficiency of both natural and artificial selection. Crossovers also form a physical link between homologous chromosomes at metaphase I which is critical for accurate chromosome segregation and fertility. The patterning of crossovers along the length of chromosomes is a highly regulated process, and our current understanding of its regulation forms the focus of this review. At the global scale, crossover patterning in plants is largely governed by the classically observed phenomena of crossover interference, crossover homeostasis and the obligatory crossover which regulate the total number of crossovers and their relative spacing. The molecular actors behind these phenomena have long remained obscure, but recent studies in plants implicate HEI10 and ZYP1 as key players in their coordination. In addition to these broad forces, a wealth of recent studies has highlighted how genomic and epigenomic features shape crossover formation at both chromosomal and local scales, revealing that crossovers are primarily located in open chromatin associated with gene promoters and terminators with low nucleosome occupancy.
10 citations
TL;DR: In this paper , the authors describe a meiotic DSB-defective mutant in barley ( Hordeum vulgare L.), which shows complete sterility due to the absence of meiotic DNA double-strand breaks, synaptonemal complex (SC), and CO formation leading to the occurrence of univalents and their unbalanced segregation into aneuploid gametes.
Abstract: Abstract Key message In barley ( Hordeum vulgare ), MTOPVIB is critical for meiotic DSB and accompanied SC and CO formation while dispensable for meiotic bipolar spindle formation. Abstract Homologous recombination during meiosis assures genetic variation in offspring. Programmed meiotic DNA double-strand breaks (DSBs) are repaired as crossover (CO) or non-crossover (NCO) during meiotic recombination. The meiotic topoisomerase VI (TopoVI) B subunit (MTOPVIB) plays an essential role in meiotic DSB formation critical for CO-recombination. More recently MTOPVIB has been also shown to play a role in meiotic bipolar spindle formation in rice and maize. Here, we describe a meiotic DSB-defective mutant in barley ( Hordeum vulgare L.). CRISPR-associated 9 (Cas9) endonuclease-generated mtopVIB plants show complete sterility due to the absence of meiotic DSB, synaptonemal complex (SC), and CO formation leading to the occurrence of univalents and their unbalanced segregation into aneuploid gametes. In HvmtopVIB plants, we also frequently found the bi-orientation of sister kinetochores in univalents during metaphase I and the precocious separation of sister chromatids during anaphase I. Moreover, the near absence of polyads after meiosis II, suggests that despite being critical for meiotic DSB formation in barley, MTOPVIB seems not to be strictly required for meiotic bipolar spindle formation.
7 citations
TL;DR: In this article , the role of the REC114/TOPOVIBL interaction in the formation of double strand break (DSB) in mice was investigated. But the authors did not identify the conserved interacting domains by structural analysis.
Abstract: Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.
6 citations
TL;DR: This study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.
Abstract: During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.
5 citations
TL;DR: In this paper , the authors show that the ASY1 remodeling complex is temporally and spatially differently assembled and that PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis.
Abstract: Abstract Chromosome axis-associated HORMA domain proteins (HORMADs), e.g. ASY1 in Arabidopsis, are crucial for meiotic recombination. ASY1, as other HORMADs, is assembled on the axis at early meiosis and depleted when homologous chromosomes synapse. Puzzlingly, both processes are catalyzed by AAA+ ATPase PCH2 together with its cofactor COMET. Here, we show that the ASY1 remodeling complex is temporally and spatially differently assembled. While PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis, PCH2 is recruited by the transverse filament protein ZYP1 and brought to the ASY1-bound COMET assuring the timely removal of ASY1 during chromosome synapsis. Since we found that the PCH2 homolog TRIP13 also binds to the ZYP1 homolog SYCP1 in mouse, we postulate that this mechanism is conserved among eukaryotes. Deleting the PCH2 binding site of ZYP1 led to a failure of ASY1 removal. Interestingly, the placement of one obligatory crossover per homologous chromosome pair, compromised by ZYP1 depletion, is largely restored in this separation-of-function zyp1 allele suggesting that crossover assurance is promoted by synapsis. In contrast, this zyp1 allele, similar to the zyp1 null mutant, showed elevated type I crossover numbers indicating that PCH2-mediated eviction of ASY1 from the axis restricts crossover formation.
5 citations
References
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TL;DR: These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity and provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.
1,727 citations
TL;DR: It is found that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro, and it is demonstrated that the best predictions can significantly reduce the time spent on guide screening.
Abstract: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (
http://crispor.org
) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.
1,256 citations
TL;DR: The current article reviews recent information on diverse aspects of chromosome morphogenesis, notably relationships between sisters, development of axial structure, and variations in chromatin status in an historical context.
Abstract: ▪ Abstract Meiotic chromosomes have been studied for many years, in part because of the fundamental life processes they represent, but also because meiosis involves the formation of homolog pairs, ...
1,206 citations
TL;DR: This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotics recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase.
Abstract: The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.
627 citations
TL;DR: It is shown that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3′-OH, indicating that the ends of a single DSB may be biochemically distinct at or before the initial processing step—much earlier than previously thought.
Abstract: DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.
570 citations