scholarly journals RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1008919
Author(s):  
Miguel Hernandez Sanchez-Rebato ◽  
Alida M. Bouatta ◽  
Maria E. Gallego ◽  
Charles I. White ◽  
Olivier Da Ines
Keyword(s):  
Homologous Recombination ◽  
Meiotic Recombination ◽  
Dna Supercoiling ◽  
Homology Search ◽  
Detectable Effect ◽  
Damage Repair ◽  
Meiotic Dsbs ◽  
Dna Translocase

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

scholarly journals RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis

2020 ◽  
Author(s):  
Miguel Hernandez Sanchez-Rebato ◽  
Alida M. Bouatta ◽  
Maria E. Gallego ◽  
Charles I. White ◽  
Olivier Da Ines
Keyword(s):  
Arabidopsis Thaliana ◽  
Homologous Recombination ◽  
Meiotic Recombination ◽  
Dna Supercoiling ◽  
Chromatin Remodelling ◽  
Homology Search ◽  
Detectable Effect ◽  
Meiotic Dsbs ◽  
Mitotic Cells

AbstractAn essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear.RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Absence of RAD54 in dmc1 mutants leads to meiotic chromosomal fragmentation. The action of RAD54 in meiotic RAD51 activity is thus downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2.RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.Author SummaryHomologous recombination is a universal pathway which repairs broken DNA molecules through the use of homologous DNA templates. It is both essential for maintenance of genome stability and for the generation of genetic diversity through sexual reproduction. A central step of the homologous recombination process is the search for and invasion of a homologous intact DNA sequence that will be used as template. This key step is catalysed by the RAD51 recombinase in somatic cells and RAD51 and DMC1 in meiotic cells, assisted by a number of associated factors. Among these, the chromatin-remodelling protein RAD54 is a required cofactor for RAD51 in mitotic cells. Understanding of its role during meiotic recombination however remains elusive. We show here that RAD54 is required for repair of meiotic double strand breaks by RAD51 in the plant Arabidopsis thaliana, and this function is downstream of the meiotic role of RAD51 in supporting the activity of DMC1. These results provide new insights into the regulation of the central step of homologous recombination in plants and very probably also other multicellular eukaryotes.

scholarly journals Genetic Studies of mei-P26 Reveal a Link Between the Processes That Control Germ Cell Proliferation in Both Sexes and Those That Control Meiotic Exchange in Drosophila

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1757-1772 ◽  
Author(s):  
Scott L Page ◽  
Kim S McKim ◽  
Benjamin Deneen ◽  
Tajia L Van Hook ◽  
R Scott Hawley
Keyword(s):  
Meiotic Recombination ◽  
Double Mutant ◽  
P Element ◽  
Genetic Studies ◽  
Germline Differentiation ◽  
Males And Females ◽  
Bag Of Marbles ◽  
Differentiation And Proliferation

Abstract We present the cloning and characterization of mei-P26, a novel P-element-induced exchange-defective female meiotic mutant in Drosophila melanogaster. Meiotic exchange in females homozygous for mei-P261 is reduced in a polar fashion, such that distal chromosomal regions are the most severely affected. Additional alleles generated by duplication of the P element reveal that mei-P26 is also necessary for germline differentiation in both females and males. To further assess the role of mei-P26 in germline differentiation, we tested double mutant combinations of mei-P26 and bag-of-marbles (bam), a gene necessary for the control of germline differentiation and proliferation in both sexes. A null mutation at the bam locus was found to act as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P261; bamΔ86/+ females is also severely decreased in comparison to mei-P261 homozygotes, indicating that bam affects the meiotic phenotype as well. These data suggest that the pathways controlling germline differentiation and meiotic exchange are related and that factors involved in the mitotic divisions of the germline may regulate meiotic recombination.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Regulation and pharmacological targeting of RAD51 in cancer

NAR Cancer ◽  
2020 ◽  
Vol 2 (3) ◽  
Author(s):  
McKenzie K Grundy ◽  
Ronald J Buckanovich ◽  
Kara A Bernstein
Keyword(s):  
Ovarian Cancer ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Patient Outcomes ◽  
Cancer Biology ◽  
Cancer Resistance ◽  
Breast And Ovarian Cancer ◽  
Resistance To Therapy ◽  
Damage Repair

Abstract Regulation of homologous recombination (HR) is central for cancer prevention. However, too little HR can increase cancer incidence, whereas too much HR can drive cancer resistance to therapy. Importantly, therapeutics targeting HR deficiency have demonstrated a profound efficacy in the clinic improving patient outcomes, particularly for breast and ovarian cancer. RAD51 is central to DNA damage repair in the HR pathway. As such, understanding the function and regulation of RAD51 is essential for cancer biology. This review will focus on the role of RAD51 in cancer and beyond and how modulation of its function can be exploited as a cancer therapeutic.

scholarly journals Ndj1, a Telomere-Associated Protein, Promotes Meiotic Recombination in Budding Yeast

2006 ◽  
Vol 26 (10) ◽  
pp. 3683-3694 ◽  
Author(s):  
Hsin-Yen Wu ◽  
Sean M. Burgess
Keyword(s):  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Homology Search ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Synaptonemal Complex Formation ◽  
Strand Invasion ◽  
Double Holliday Junction ◽  
Meiosis I

ABSTRACT Dynamic telomere repositioning is a prominent feature of meiosis. Deletion of a telomere-associated protein, Ndj1, results in the failure of both attachment and clustering of telomeres at the nuclear envelope and delays several landmarks of meiosis I, such as pairing, synaptonemal complex formation, and timing of the meiosis I division. We explored the role of Ndj1 in meiotic recombination, which occurs through the formation and repair of programmed double-strand breaks. The ndj1Δ mutation allows for the formation of the first detectable strand invasion intermediate (i.e., single-end invasion) with wild-type kinetics; however, it confers a delay in the formation of the double-Holliday junction intermediate and both crossover and noncrossover products. These results challenge the widely held notion that clustering of telomeres in meiosis promotes the ability of homologous chromosomes to find one another in budding Saccharomyces cerevisiae. We propose that an Ndj1-dependent function is critical for stabilizing analogous strand invasion intermediates that exist in two separate branches of the bifurcated pathway, leading to either noncrossover or crossover formation. These findings provide a link between telomere dynamics and a distinct mechanistic step of meiotic recombination that follows the homology search.

scholarly journals Schizosaccharomyces pombeRdh54 (TID1) Acts with Rhp54 (RAD54) to Repair Meiotic Double-Strand Breaks

2003 ◽  
Vol 14 (11) ◽  
pp. 4707-4720 ◽  
Author(s):  
Michael G. Catlett ◽  
Susan L. Forsburg
Keyword(s):  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Cell Analysis ◽  
Yeast Two Hybrid ◽  
Spore Viability ◽  
Strand Breaks ◽  
Functional Homology ◽  
Damage Repair ◽  
Two Hybrid

We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Δ shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Δ single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Δ rhp54Δ double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Δ rhp54Δ asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.

scholarly journals ATR is required to complete meiotic recombination in mice

2017 ◽  
Author(s):  
Sarai Pacheco ◽  
Andros Maldonado-Linares ◽  
Marina Marcet-Ortega ◽  
Cristina Rojas ◽  
Ana Martínez-Marchal ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Meiotic Prophase ◽  
Protein A ◽  
Homology Search ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Molecular Features ◽  
Chromosome Synapsis ◽  
Meiotic Dsbs

ABSTRACTPrecise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

scholarly journals The tumor suppressor BRCA1/BARD1 complex localizes to the synaptonemal complex and regulates recombination under meiotic dysfunction in Caenorhabditis elegans

2018 ◽  
Author(s):  
Qianyan Li ◽  
Takamune T. Saito ◽  
Alison J. Deshong ◽  
Marina Martinez Garcia ◽  
Saravanapriah Nadarajan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Caenorhabditis Elegans ◽  
Tumor Suppressor ◽  
Homologous Recombination ◽  
Synaptonemal Complex ◽  
Meiotic Recombination ◽  
Dna Damage Repair ◽  
Chromosome Synapsis ◽  
Brca1 Mutations ◽  
Damage Repair

AbstractBreast cancer susceptibility gene 1(BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. In Caenorhabditis elegans BRCA1/BRC-1 and BARD1/BRD-1 orthologs are not essential, but function in DNA damage repair and homologous recombination, as well as in meiosis. In proliferating germ cells and in early meiotic prophase, BRC-1 and BRD-1 are nucleoplasmic, with enrichment at foci that partially overlap with the recombinase RAD-51. In mid-pachytene, BRC-1 and BRD-1 are observed on tracks, before concentrating to the short arms of bivalents, co-localizing with a central region component of the synaptonemal complex. We found that BRD-1 is essential for BRC-1 to associate with chromatin and the synaptonemal complex, but BRC-1 is not required for BRD-1 localization; the complex fails to properly localize in the absence of either meiotic recombination or chromosome synapsis. Inactivation of BRC-1/BRD-1 enhances the embryonic lethality of mutants that perturb chromosome synapsis and crossover recombination, suggesting that BRC-1/BRD-1 plays an important role in monitoring recombination in the context of the synaptonemal complex. We discovered that BRC-1/BRD-1 stabilizes the RAD51 filament when the formation of a crossover-intermediate is disrupted. Further, in the absence of BRC-1/BRD-1 crossover distribution is altered, and under meiotic dysfunction, crossover numbers are perturbed. Together, our studies indicate that BRC-1/BRD-1 localizes to the synaptonemal complex where it serves a checkpoint function to monitor and modulate meiotic recombination.Project SummaryOur genomes are passed down from one generation to the next through the specialized cell division program of meiosis. Meiosis is highly regulated to coordinate both the large scale chromosomal and fine scale DNA events to ensure fidelity. We analyzed the role of the tumor suppressor BRCA1/BARD1 complex in meiosis in the worm, Caenorhabditis elegans. We find that BRCA1/BARD1 localizes dynamically to the proteinaeous structure that aligns maternal and paternal chromosomes, where it regulates crossover recombination. Although BRCA1/BARD1 mutants have only subtle meiotic defects, we show that this complex plays a critical role in meiotic recombination when meiosis is perturbed. These results highlight the complexity of ensuring accurate transmission of the genome and uncover the requirement for this conserved complex in meiosis. As women carrying BRCA1 mutations with no indication of cancer have fertility defects, our results provide insight into why BRCA1 mutations impact reproductive success.

scholarly journals RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis

2020 ◽  
Author(s):  
Adam Hefel ◽  
Nicholas Cronin ◽  
Kailey Harrel ◽  
Pooja Patel ◽  
Maria Spies ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Homologous Recombination ◽  
Meiotic Recombination ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
Filament Formation ◽  
C Elegans ◽  
Trimeric Complex ◽  
Meiotic Dsbs

AbstractReplication Protein A (RPA) is critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes for RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determine that RPA-2 is critical for germline replication, and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for replication, contradictory to what is seen in other organisms, that require both subunits. In the germline, both RPA-1/2 and RPA-1/4 complexes form, but RPA-1/4 is less abundant and its formation is repressed by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibit RAD-51 filament formation and promotes apoptosis on a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans.

scholarly journals The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination.

Genetics ◽  
1995 ◽  
Vol 141 (1) ◽  
pp. 49-59
Author(s):  
B Rockmill ◽  
J A Engebrecht ◽  
H Scherthan ◽  
J Loidl ◽  
G S Roeder
Keyword(s):  
Meiotic Recombination ◽  
Microscopic Analysis ◽  
Primary Defect ◽  
Electron Microscopic ◽  
Strand Breaks ◽  
Meiotic Chromosomes ◽  
Chromosome Synapsis ◽  
Damage Repair

Abstract Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.

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