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1.
Cell ; 184(24): 5970-5984.e18, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34793701

RESUMO

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.


Assuntos
Deleção de Genes , Duplicação Gênica , Células Germinativas/metabolismo , Recombinação Genética/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Cromátides/metabolismo , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Quebras de DNA de Cadeia Dupla , DNA Circular/genética , Feminino , Genoma , Haplótipos/genética , Recombinação Homóloga/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mutagênese Insercional/genética , Mutação/genética
2.
Cell Cycle ; 17(10): 1155-1172, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29963942

RESUMO

DNA double-strand breaks (DSBs) generated by the SPO11 protein initiate meiotic recombination, an essential process for successful chromosome segregation during gametogenesis. The activity of SPO11 is controlled by multiple factors and regulatory mechanisms, such that the number of DSBs is limited and DSBs form at distinct positions in the genome and at the right time. Loss of this control can affect genome integrity or cause meiotic arrest by mechanisms that are not fully understood. Here we focus on the DSB-responsive kinase ATM and its functions in regulating meiotic DSB numbers and distribution. We review the recently discovered roles of ATM in this context, discuss their evolutionary conservation, and examine future research perspectives.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Meiose , Animais , Humanos , Modelos Biológicos
3.
DNA Repair (Amst) ; 35: 137-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26519827

RESUMO

The resection of 5'-DNA ends at a double-strand break (DSB) is an essential step in recombinational repair, as it exposes 3' single-stranded DNA (ssDNA) tails for interaction with a repair template. In mitosis, Exo1 and Sgs1 have a conserved function in the formation of long ssDNA tails, whereas this step in the processing of programmed meiotic DSBs is less well-characterized across model organisms. In budding yeast, which has been most intensely studied in this respect, Exo1 is a major meiotic nuclease. In addition, it exerts a nuclease-independent function later in meiosis in the conversion of DNA joint molecules into ZMM-dependent crossovers. In order to gain insight into the diverse meiotic roles of Exo1, we investigated the effect of Exo1 deletion in the ciliated protist Tetrahymena. We found that Exo1 together with Mre11, but without the help of Sgs1, promotes meiotic DSB end resection. Resection is completely eliminated only if both Mre11 and Exo1 are missing. This is consistent with the yeast model where Mre11 promotes resection in the 3'-5' direction and Exo1 in the opposite 5'-3' direction. However, while the endonuclease activity of Mre11 is essential to create an entry site for exonucleases and hence to start resection in budding yeast, Tetrahymena Exo1 is able to create single-stranded DNA in the absence of Mre11. Excluding a possible contribution of the Mre11 cofactor Sae2 (Com1) as an autonomous endonuclease, we conclude that there exists another unknown nuclease that initiates DSB processing in Tetrahymena. Consistent with the absence of the ZMM crossover pathway in Tetrahymena, crossover formation is independent of Exo1.


Assuntos
Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Protozoários/metabolismo , Reparo de DNA por Recombinação , Tetrahymena thermophila/genética , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Endonucleases/química , Exodesoxirribonucleases/genética , Técnicas de Inativação de Genes , Meiose , Proteínas de Protozoários/genética , Tetrahymena thermophila/enzimologia
4.
Genetics ; 198(3): 983-93, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25217051

RESUMO

ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.


Assuntos
Troca Genética , Meiose , Proteínas de Protozoários/metabolismo , Complexo Sinaptonêmico/metabolismo , Tetrahymena/citologia , Tetrahymena/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Protozoário/metabolismo , Técnicas de Inativação de Genes , Mutação/genética , Filogenia , Homologia de Sequência de Aminoácidos
5.
Nucleic Acids Res ; 41(20): 9296-309, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23935123

RESUMO

Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.


Assuntos
Endodesoxirribonucleases/fisiologia , Meiose/genética , RecQ Helicases/fisiologia , Recombinases/fisiologia , Recombinação Genética , Núcleo Celular/enzimologia , Cromátides , Segregação de Cromossomos , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Mutação , Interferência de RNA , RecQ Helicases/análise , RecQ Helicases/antagonistas & inibidores , Recombinases/análise , Recombinases/antagonistas & inibidores , Complexo Sinaptonêmico , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genética
6.
PLoS Genet ; 9(3): e1003418, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23555314

RESUMO

The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Meiose/genética , Mitose/genética , Tetrahymena , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Dano ao DNA , Reparo do DNA/genética , Endopeptidases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Separase , Tetrahymena/citologia , Tetrahymena/genética , Coesinas
7.
J Cell Sci ; 125(Pt 23): 5873-80, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22976299

RESUMO

In order to form crossovers and to undergo reductional segregation during meiosis, homologous chromosomes must pair. In Tetrahymena, meiotic prophase nuclei elongate immensely, and, within the elongated nucleus, chromosomes are arranged with telomeres assembled at one pole and centromeres at the opposite pole. This organisation is an exaggerated form of the bouquet, a meiotic chromosome arrangement that is widely conserved among eukaryotes. We show that centromere function is crucial for the formation of Tetrahymena's stretched bouquet and, thereby, for homologue pairing. This finding adds to previous reports of the importance of centromeres in chromosome pairing in budding yeast and in Drosophila. Tetrahymena's bouquet is an ataxia telangiectasia- and RAD3-related (ATR)-dependent meiotic DNA damage response that is triggered by meiotic DNA double-strand breaks (DSBs), suggesting that the bouquet is needed for DSB repair. However, in the present study we show that although homologous pairing is impeded in the absence of the bouquet, DSB repair takes place nevertheless. Moreover, recombinational DSB repair, as monitored by bromodeoxyuridine incorporation, takes place only after exit from the bouquet stage. Therefore, we conclude that the bouquet is not required for DSB repair per se, but may be necessary for the alignment of homologous loci in order to promote homologous crossovers over alternative repair pathways.


Assuntos
Centrômero/genética , Cromossomos/genética , Recombinação Genética/genética , Tetrahymena/genética , Pareamento Cromossômico/genética , Meiose/genética
8.
PLoS Genet ; 7(3): e1001359, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21483758

RESUMO

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3' single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA-strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB-dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Meiose/genética , Rad51 Recombinase/fisiologia , Tetrahymena/genética , Proteínas de Ciclo Celular/genética , Troca Genética , Rad51 Recombinase/genética , Recombinação Genética , Tetrahymena/citologia , Tetrahymena/enzimologia
9.
Chromosoma ; 119(5): 505-18, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20422424

RESUMO

Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.


Assuntos
Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Meiose , Proteínas de Protozoários/metabolismo , Tetrahymena/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Troca Genética , Dano ao DNA , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Protozoários/genética , Recombinação Genética , Tetrahymena/citologia , Tetrahymena/metabolismo
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