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 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 ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

2021 ◽  
Vol 118 (14) ◽  
pp. e2021671118
Author(s):  
Martin G. France ◽  
Janina Enderle ◽  
Sarah Röhrig ◽  
Holger Puchta ◽  
F. Chris H. Franklin ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Synaptonemal Complex ◽  
Class Ii ◽  
Class I ◽  
Crossing Over ◽  
Wild Type ◽  
Homologous Chromosomes ◽  
Virtual Absence ◽  
Meiotic Progression ◽  
Null Mutants

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis. Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

scholarly journals ATP-Dependent Recruitment of Export Factor Aly/REF onto Intronless mRNAs by RNA Helicase UAP56

2007 ◽  
Vol 28 (2) ◽  
pp. 601-608 ◽  
Author(s):  
Ichiro Taniguchi ◽  
Mutsuhito Ohno
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Adaptor Proteins ◽  
Rna Helicase ◽  
Specific Protein ◽  
Binding Activity ◽  
Exon Junction Complex ◽  
Dependent Manner ◽  
Insight Into

ABSTRACT Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

scholarly journals Seventeen complementation groups of mutations decreasing meiotic recombination in Schizosaccharomyces pombe.

Genetics ◽  
1992 ◽  
Vol 130 (2) ◽  
pp. 251-262 ◽  
Author(s):  
L C De Veaux ◽  
N A Hoagland ◽  
G R Smith
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Meiotic Recombination ◽  
Class Ii ◽  
Class I ◽  
Intragenic Recombination ◽  
Class Iii ◽  
Multiple Alleles ◽  
Fold Reduction ◽  
Complementation Groups ◽  
Rec Genes

Abstract We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.

scholarly journals Multiple Domains of Fission Yeast Cdc19p (MCM2) Are Required for Its Association with the Core MCM Complex

1998 ◽  
Vol 9 (7) ◽  
pp. 1833-1845 ◽  
Author(s):  
Daniel A. Sherman ◽  
Sally G. Pasion ◽  
Susan L. Forsburg
Keyword(s):  
Fission Yeast ◽  
Protein Complexes ◽  
Gel Filtration ◽  
The Core ◽  
Eukaryotic Dna ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Multiple Domains ◽  
Replication Factors

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.

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.

Electron microscopy analysis of degenerating pancreatic ß cells in transgenic mice expressing the MHC Class I antigen Dd

1990 ◽  
Vol 48 (3) ◽  
pp. 548-549
Author(s):  
T. A. Stewart ◽  
D. Liggitt ◽  
S. Pitts ◽  
L. Martin ◽  
M. Siegel ◽  
...  
Keyword(s):  
Type I Diabetes ◽  
Disease Process ◽  
Class Ii ◽  
Class I ◽  
Type I ◽  
Aberrant Expression ◽  
Mhc Molecules ◽  
Endogenous Insulin ◽  
Class Ii Mhc ◽  
Ifn Γ

Insulin-dependant (Type I) diabetes mellitus (IDDM) is a metabolic disorder resulting from the lack of endogenous insulin secretion. The disease is thought to result from the autoimmune mediated destruction of the insulin producing ß cells within the islets of Langerhans. The disease process is probably triggered by environmental agents, e.g. virus or chemical toxins on a background of genetic susceptibility associated with particular alleles within the major histocompatiblity complex (MHC). The relation between IDDM and the MHC locus has been reinforced by the demonstration of both class I and class II MHC proteins on the surface of ß cells from newly diagnosed patients as well as mounting evidence that IDDM has an autoimmune pathogenesis. In 1984, a series of observations were used to advance a hypothesis, in which it was suggested that aberrant expression of class II MHC molecules, perhaps induced by gamma-interferon (IFN γ) could present self antigens and initiate an autoimmune disease. We have tested some aspects of this model and demonstrated that expression of IFN γ by pancreatic ß cells can initiate an inflammatory destruction of both the islets and pancreas and does lead to IDDM.

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