scholarly journals A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

2019 ◽  
Author(s):  
Diedre Reitz ◽  
Jennifer Grubb ◽  
Douglas K. Bishop
Keyword(s):  
Meiotic Recombination ◽  
Genome Rearrangements ◽  
Accessory Protein ◽  
Filament Formation ◽  
Single Stranded Dna ◽  
Homologous Chromosomes ◽  
Sister Chromatids ◽  
Accessory Factor ◽  
Accessory Factors ◽  
Insight Into

AbstractDuring meiosis, homologous recombination repairs programmed DNA double-stranded breaks (DSBs). Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. We also provide evidence that Mei5-Sae3 promotes Dmc1 filament formation specifically on single-stranded DNA. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister (IS) joint molecules intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1 (Dmc1-WT). Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filaments lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.Author SummaryDuring meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover (CO). Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements.

scholarly journals A dCas9-Based System Identifies a Central Role for Ctf19 in Kinetochore-Derived Suppression of Meiotic Recombination

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 395-408
Author(s):  
Lisa-Marie Kuhl ◽  
Vasso Makrantoni ◽  
Sarah Recknagel ◽  
Animish N. Vaze ◽  
Adele L. Marston ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Genome Stability ◽  
Meiotic Chromosome ◽  
Homologous Chromosomes ◽  
Experimental Platform ◽  
Chromosome Missegregation ◽  
Link Type ◽  
Insight Into

In meiosis, crossover (CO) formation between homologous chromosomes is essential for faithful segregation. However, misplaced meiotic recombination can have catastrophic consequences on genome stability. Within pericentromeres, COs are associated with meiotic chromosome missegregation. In organisms ranging from yeast to humans, pericentromeric COs are repressed. We previously identified a role for the kinetochore-associated Ctf19 complex (Ctf19c) in pericentromeric CO suppression. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits. Using this approach, we query sufficiency in meiotic CO suppression, and identify Ctf19 as a mediator of kinetochore-associated CO control. The effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues important for the recruitment of the Scc2-Scc4 cohesin regulator. This work provides insight into kinetochore-derived control of meiotic recombination. We establish an experimental platform to investigate and manipulate meiotic CO control. This platform can easily be adapted in order to investigate other aspects of chromosome biology.

scholarly journals A dCas9/CRISPR-based targeting system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination

2020 ◽  
Author(s):  
Lisa-Marie Kuhl ◽  
Vasso Makrantoni ◽  
Sarah Recknagel ◽  
Animish N. Vaze ◽  
Adele L. Marston ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Genome Stability ◽  
Budding Yeast ◽  
Homologous Chromosomes ◽  
Experimental Platform ◽  
Chromosome Missegregation ◽  
Meiotic Crossover ◽  
Insight Into

AbstractIn meiosis, crossover formation between homologous chromosomes is essential for faithful segregation. However, improperly controlled or placed meiotic recombination can have catastrophic consequences on genome stability. Specifically, within centromeres and surrounding regions (i.e. pericentromeres), crossovers are associated with chromosome missegregation and developmental aneuploidy. In organisms ranging from yeast to humans, crossovers are repressed within (peri)centromeric regions. We previously identified a key role for the multi-subunit, kinetochore-associated Ctf19 complex (Ctf19c; the budding yeast equivalent of the human CCAN) in regulating pericentromeric crossover formation. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits to a non-centromeric locus during meiosis. Using this approach, we query sufficiency in meiotic crossover suppression, and identify Ctf19 (the budding yeast homologue of vertebrate CENP-P) as a central mediator of kinetochore-associated crossover control. We show that the effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues known to be important for the recruitment of the Scc2-Scc4 cohesin regulator to kinetochores. We thus reveal a crucial determinant that links kinetochores to meiotic recombinational control. This work provides insight into localized control of meiotic recombination. Furthermore, our approach establishes a dCas9/CRISPR-based experimental platform that can be utilized to investigate and locally manipulate meiotic crossover control. This platform can easily be adapted in order to investigate other aspects of localized chromosome biology.

scholarly journals Meiotic MCM proteins promote and inhibit crossovers during meiotic recombination

2018 ◽  
Author(s):  
Michaelyn Hartmann ◽  
Kathryn P. Kohl ◽  
Jeff Sekelsky ◽  
Talia Hatkevich
Keyword(s):  
Meiotic Recombination ◽  
Protein Complexes ◽  
Class Ii ◽  
Specific Protein ◽  
Class I ◽  
Homologous Chromosomes ◽  
N Terminus ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Insight Into

AbstractCrossover formation as a result of meiotic recombination is vital for proper segregation of homologous chromosomes at the end of meiosis I. In many organisms, crossovers are generated through two crossover pathways: Class I and Class II. To ensure accurate crossover formation, meiosis-specific protein complexes regulate the degree in which each pathway is used. One such complex is the mei-MCM complex, which contains MCM (mini-chromosome maintenance) and MCM-like proteins REC (ortholog of Mcm8), MEI-217, and MEI-218, collectively called the mei-MCM complex. The mei-MCM complex genetically promotes Class I crossovers and inhibits Class II crossovers in Drosophila, but it is unclear how individual mei-MCM proteins contribute to crossover regulation. In this study, we perform genetic analyses to understand how specific regions and motifs of mei-MCM proteins contribute to Class I and II crossover formation and distribution. Our analyses show that the long, disordered N-terminus of MEI-218 is dispensable for crossover formation, and that mutations that disrupt REC’s Walker A and B motifs differentially affect Class I and Class II crossover formation. In Rec Walker A mutants, Class I crossovers exhibit no change, but Class II crossovers are increased. However, in rec Walker B mutants, Class I crossovers are severely impaired, and Class II crossovers are increased. These results suggest that REC may form multiple complexes that exhibit differential REC-dependent ATP binding and hydrolyzing requirements. These results provide genetic insight into the mechanisms through which mei-MCM proteins promote Class I crossovers and inhibit Class II crossovers.

scholarly journals Partner Choice during Meiosis Is Regulated by Hop1-promoted Dimerization of Mek1

2005 ◽  
Vol 16 (12) ◽  
pp. 5804-5818 ◽  
Author(s):  
Hengyao Niu ◽  
Lihong Wan ◽  
Bridget Baumgartner ◽  
Dana Schaefer ◽  
Josef Loidl ◽  
...  
Keyword(s):  
Point Mutation ◽  
Sister Chromatid ◽  
Meiotic Recombination ◽  
Partner Choice ◽  
Dsb Repair ◽  
Spore Viability ◽  
Homologous Chromosomes ◽  
Chromosome Synapsis ◽  
Sister Chromatids ◽  
Specific Proteins

Meiotic recombination differs from mitotic recombination in that DSBs are repaired using homologous chromosomes, rather than sister chromatids. This change in partner choice is due in part to a barrier to sister chromatid repair (BSCR) created by the meiosis-specific kinase, Mek1, in a complex with two other meiosis-specific proteins, Hop1 and Red1. HOP1 contains two functional domains, called the N and C domains. Analysis of a point mutation that specifically inactivates the C domain (hop1-K593A) reveals that the N domain is sufficient for Hop1 localization to chromosomes and for Red1 and Hop1 interactions. The C domain is needed for spore viability, for chromosome synapsis, and for preventing DMC1-independent DSB repair, indicating it plays a role in the BSCR. All of the hop1-K593A phenotypes can be bypassed by fusion of ectopic dimerization domains to Mek1, suggesting that the function of the C domain is to promote Mek1 dimerization. Hop1 is a DSB-dependent phosphoprotein, whose phosphorylation requires the presence of the C domain, but is independent of MEK1. These results suggest a model in which Hop1 phosphorylation in response to DSBs triggers dimerization of Mek1 via the Hop1 C domain, thereby enabling Mek1 to phosphorylate target proteins that prevent repair of DSBs by sister chromatids.

Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 947-957 ◽  
Author(s):  
John G Jelesko ◽  
Kristy Carter ◽  
Whitney Thompson ◽  
Yuki Kinoshita ◽  
Wilhelm Gruissem
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Meiotic Recombination ◽  
Sequence Similarity ◽  
Specific Gene ◽  
High Sequence Similarity ◽  
Paralogous Genes ◽  
Chimeric Genes ◽  
Unequal Recombination ◽  
Sister Chromatids

Abstract Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

scholarly journals Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

1999 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Stéphane Brunet ◽  
Angélica Santa Maria ◽  
Philippe Guillaud ◽  
Denis Dujardin ◽  
Jacek Z. Kubiak ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Spindle ◽  
Homologous Chromosomes ◽  
Mouse Oocytes ◽  
Sister Chromatids ◽  
M Phase ◽  
Continuous Presence ◽  
Set Up ◽  
Polar Body Extrusion ◽  
Reductional Division

During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion.

Chromosome organization of the protozoan Trypanosoma brucei

1990 ◽  
Vol 10 (11) ◽  
pp. 6079-6083
Author(s):  
K Gottesdiener ◽  
J Garciá-Anoveros ◽  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Dna Sequences ◽  
Chromosomal Location ◽  
Karyotype Analysis ◽  
Enzyme Analysis ◽  
Chromosome Organization ◽  
Restriction Enzyme Analysis ◽  
Valuable Insight ◽  
Homologous Chromosomes ◽  
Insight Into

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determining the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.

scholarly journals Inverted meiosis: an alternative way of chromosome segregation for reproduction

2020 ◽  
Vol 52 (7) ◽  
pp. 702-707 ◽  
Author(s):  
Wenzhu Li ◽  
Xiangwei He
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Chromosome Structure ◽  
Adaptive Strategy ◽  
Homologous Chromosomes ◽  
Working Model ◽  
Sister Chromatids ◽  
Holocentric Chromosome ◽  
Inverted Order

Abstract Canonical meiosis is characterized by two sequential rounds of nuclear divisions following one round of DNA replication—reductional segregation of homologous chromosomes during the first division and equational segregation of sister chromatids during the second division. Meiosis in an inverted order of two nuclear divisions—inverted meiosis has been observed in several species with holocentromeres as an adaptive strategy to overcome the obstacle in executing a canonical meiosis due to the holocentric chromosome structure. Recent findings of co-existence of inverted and canonical meiosis in two monocentric organisms, human and fission yeast, suggested that inverted meiosis could be common and also lead to the puzzle regarding the mechanistic feasibility for executing two meiosis programs simultaneously. Here, we discuss apparent conflicts for concurrent canonical meiosis and inverted meiosis. Furthermore, we attempt to provide a working model that may be compatible for both forms of meiosis.

scholarly journals Three-dimensional Ultrastructure of Saccharomyces cerevisiae Meiotic Spindles

2005 ◽  
Vol 16 (3) ◽  
pp. 1178-1188 ◽  
Author(s):  
Mark Winey ◽  
Garry P. Morgan ◽  
Paul D. Straight ◽  
Thomas H. Giddings ◽  
David N. Mastronarde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Meiotic Chromosome ◽  
Three Dimensional ◽  
Structural Basis ◽  
Mitotic Spindles ◽  
Yeast Saccharomyces Cerevisiae ◽  
Homologous Chromosomes ◽  
Sister Chromatids ◽  
Single Nucleus ◽  
Meiotic Spindles

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.

scholarly journals Extensive Loss of Heterozygosity Is Suppressed during Homologous Repair of Chromosomal Breaks

2003 ◽  
Vol 23 (2) ◽  
pp. 733-743 ◽  
Author(s):  
Jeremy M. Stark ◽  
Maria Jasin
Keyword(s):  
Loss Of Heterozygosity ◽  
Meiotic Recombination ◽  
Mammalian Cells ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Human Populations ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Homologous Chromosomes

ABSTRACT Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 × 10−5; however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.

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