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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445523

RESUMO

The accurate segregation of chromosomes is essential for the survival of organisms and cells. Mistakes can lead to aneuploidy, tumorigenesis and congenital birth defects. The spindle assembly checkpoint ensures that chromosomes properly align on the spindle, with sister chromatids attached to microtubules from opposite poles. Here, we review how tension is used to identify and selectively destabilize incorrect attachments, and thus serves as a trigger of the spindle assembly checkpoint to ensure fidelity in chromosome segregation. Tension is generated on properly attached chromosomes as sister chromatids are pulled in opposing directions but resisted by centromeric cohesin. We discuss the role of the Aurora B kinase in tension-sensing and explore the current models for translating mechanical force into Aurora B-mediated biochemical signals that regulate correction of chromosome attachments to the spindle.


Assuntos
Aurora Quinase B/metabolismo , Cromátides/metabolismo , Cinetocoros/metabolismo , Animais , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular
2.
Artigo em Inglês | MEDLINE | ID: mdl-34266623

RESUMO

We evaluated the sensitivity of the chromosomal aberration (CA) and mitotic index (MI) assays on peripheral blood lymphocytes (PBLs) of Caiman latirostris, following ex vivo exposure to the alkylating agent, MMS. Two concentrations of MMS were tested in cultured peripheral blood. Relative to controls, MMS exposure reduced the number of metaphases observed, but both the numbers of cells with MN and the percentages of aberrant metaphases increased. The types of CA identified were chromosome and chromatid breaks, chromosomal rearrangements, monosomies, and nullisomies, with significantly higher values in the MMS-exposed groups. The incorporation of the MI and CA tests in C. latirostris can provide information on damage caused by xenobiotic exposures.


Assuntos
Alquilantes/toxicidade , Jacarés e Crocodilos/genética , Biomarcadores/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Animais , Células Cultivadas , Cromátides/efeitos dos fármacos , Cromátides/genética , Cromossomos/genética , Linfócitos/efeitos dos fármacos , Testes para Micronúcleos/métodos , Mitose/genética , Índice Mitótico/métodos
3.
Artigo em Inglês | MEDLINE | ID: mdl-34266628

RESUMO

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.


Assuntos
Cromátides/genética , Reparo do DNA por Junção de Extremidades/genética , DNA/genética , Recombinação Homóloga/genética , Reparo de DNA por Recombinação/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Fase G2/genética , Poli(ADP-Ribose) Polimerase-1/genética , Radiação Ionizante
4.
Front Public Health ; 9: 675095, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34123995

RESUMO

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.


Assuntos
Cromátides , Reparo do DNA , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Tolerância a Radiação
5.
Nat Commun ; 12(1): 2917, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006877

RESUMO

Topoisomerase II (topo II) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood, however, mechanistically how this enzyme regulates this process. In an attempt to further refine the reconstitution assay, we have found that chromosomal binding of Xenopus laevis topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD). Enzymological assays using circular DNA substrates supports the idea that topo IIα first resolves inter-chromatid entanglements to drive individualization and then generates intra-chromatid entanglements to promote thickening. Importantly, only the latter process requires the CTD. By using frog egg extracts, we also show that the CTD contributes to proper formation of nucleosome-depleted chromatids by competing with a linker histone for non-nucleosomal DNA. Our results demonstrate that topo IIα utilizes its CTD to deliver the enzymatic core to crowded environments created during mitotic chromatid assembly, thereby fine-tuning this process.


Assuntos
Cromátides/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Sítios de Ligação/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromátides/genética , Segregação de Cromossomos/genética , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , Feminino , Histonas/metabolismo , Masculino , Mitose/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Espermatozoides/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética
6.
Life Sci ; 277: 119556, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33945829

RESUMO

OBJECTIVE: Poly(ADP-ribose) polymerase1 (PARP1) interacts and poly(ADP-ribosyl)ates telomere repeat binding factor 2 (TRF2), which acts as a platform to recruit a large number of proteins at the telomere. Since the discovery of TRF2-SLX4 interaction, SLX4 is becoming the key player in telomere length (TL) maintenance and repair by telomere sister chromatid exchange (T-SCE). Defective TL maintenance pathway results in a spectrum of diseases called telomeropathies like dyskeratosis congenita, aplastic anemia, fanconi anemia, cancer. We aimed to study the role of SLX4 and PARP1 on each other's telomere localization, T-SCE, and TL maintenance in human telomerase-negative osteosarcoma U2OS cells to understand some of the molecular mechanisms of telomere homeostasis. MATERIALS AND METHODS: We checked the role of SLX4 and PARP1 on each other's telomere localization by telomere immunofluorescence. We have cloned full-length wild-type and catalytically inactive mutant PARP1 to understand the role of poly(ADP-ribosyl)ation reaction by PARP1 in telomere length homeostasis. TL of U2OS cells was measured by Q-FISH. T-SCE was measured by Telomere-FISH. KEY FINDINGS: We observed that SLX4 has no role in the telomere localization of PARP1. However, reduced localization of SLX4 at undamaged and damaged telomere upon PARP1 depletion was reversed by overexpression of exogenous wild-type PARP1 but not by overexpression of catalytically inactive mutant PARP1. PARP1 depletion synergized SLX4 depletion-mediated reduction of T-SCE. Furthermore, SLX4 depletion elongated TL, and combined insufficiency of SLX4 with PARP1 further elongated TL. CONCLUSION: So, PARP1 controls SLX4 recruitment at telomere by poly(ADP-ribosyl)ation reaction, thereby regulating SLX4-mediated T-SCE and TL homeostasis.


Assuntos
Poli(ADP-Ribose) Polimerase-1/metabolismo , Recombinases/metabolismo , Troca de Cromátide Irmã/fisiologia , Linhagem Celular Tumoral , Cromátides/metabolismo , Cromátides/fisiologia , Reparo do DNA , Homeostase , Humanos , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Recombinases/genética , Recombinases/fisiologia , Telomerase/metabolismo , Telômero/fisiologia , Homeostase do Telômero/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
7.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33929514

RESUMO

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition.


Assuntos
Proteínas de Transporte/genética , Segregação de Cromossomos/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Cromátides/genética , Humanos , Cinetocoros , Mitose/genética , Proteína Fosfatase 2/genética , Saccharomyces cerevisiae/genética , Fuso Acromático/genética , Sumoilação/genética
8.
Int J Mol Sci ; 22(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917542

RESUMO

More than 70% of eukaryotic proteins are regulated by phosphorylation. However, the mechanism of dephosphorylation that counteracts phosphorylation is less studied. Phosphatases are classified into 104 distinct groups based on substrate-specific features and the sequence homologies in their catalytic domains. Among them, dual-specificity phosphatases (DUSPs) that dephosphorylate both phosphoserine/threonine and phosphotyrosine are important for cellular homeostasis. Ssu72 is a newly studied phosphatase with dual specificity that can dephosphorylate both phosphoserine/threonine and phosphotyrosine. It is important for cell-growth signaling, metabolism, and immune activation. Ssu72 was initially identified as a phosphatase for the Ser5 and Ser7 residues of the C-terminal domain of RNA polymerase II. It prefers the cis configuration of the serine-proline motif within its substrate and regulates Pin1, different from other phosphatases. It has recently been reported that Ssu72 can regulate sister chromatid cohesion and the separation of duplicated chromosomes during the cell cycle. Furthermore, Ssu72 appears to be involved in the regulation of T cell receptor signaling, telomere regulation, and even hepatocyte homeostasis in response to a variety of stress and damage signals. In this review, we aim to summarize various functions of the Ssu72 phosphatase, their implications in diseases, and potential therapeutic indications.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Animais , Cromátides/genética , Cromátides/metabolismo , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Humanos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosfoproteínas Fosfatases/genética , Domínios Proteicos , RNA Polimerase II/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo
9.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802105

RESUMO

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Replicação do DNA/fisiologia , Fase G1/fisiologia , Telófase/fisiologia , Animais , Humanos
10.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669056

RESUMO

Warsaw breakage syndrome (WABS) is a genetic disorder characterized by sister chromatid cohesion defects, growth retardation, microcephaly, hearing loss and other variable clinical manifestations. WABS is due to biallelic mutations of the gene coding for the super-family 2 DNA helicase DDX11/ChlR1, orthologous to the yeast chromosome loss protein 1 (Chl1). WABS is classified in the group of "cohesinopathies", rare hereditary diseases that are caused by mutations in genes coding for subunits of the cohesin complex or protein factors having regulatory roles in the sister chromatid cohesion process. In fact, among the cohesion regulators, an important player is DDX11, which is believed to be important for the functional coupling of DNA synthesis and cohesion establishment at the replication forks. Here, we will review what is known about the molecular and cellular functions of human DDX11 and its role in WABS etiopathogenesis, even in light of recent findings on the role of cohesin and its regulator network in promoting chromatin loop formation and regulating chromatin spatial organization.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Doenças Raras/metabolismo , Anormalidades Múltiplas/genética , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromátides/patologia , Cromatina/patologia , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , RNA Helicases DEAD-box/genética , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Mutação , Filogenia , Doenças Raras/congênito , Doenças Raras/enzimologia , Doenças Raras/fisiopatologia
11.
Gene ; 784: 145584, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33753149

RESUMO

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1mWW and Eco1mGRK), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Sítios de Ligação , Cromossomos Fúngicos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
12.
Trends Biochem Sci ; 46(4): 255-257, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33593592

RESUMO

Understanding how genomes are spatially organized is central to many aspects of cell biology. However, it has been difficult to study the relationships between sister chromatids because sequencing-based techniques such as Hi-C could not distinguish identical sister DNAs. Here, I discuss recent developments that provide insights into sister chromatid organization.


Assuntos
Cromátides , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA , Replicação do DNA
13.
Molecules ; 26(3)2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494466

RESUMO

Amsacrine, an anticancer drug first synthesised in 1970 by Professor Cain and colleagues, showed excellent preclinical activity and underwent clinical trial in 1978 under the auspices of the US National Cancer Institute, showing activity against acute lymphoblastic leukaemia. In 1984, the enzyme DNA topoisomerase II was identified as a molecular target for amsacrine, acting to poison this enzyme and to induce DNA double-strand breaks. One of the main challenges in the 1980s was to determine whether amsacrine analogues could be developed with activity against solid tumours. A multidisciplinary team was assembled in Auckland, and Professor Denny played a leading role in this approach. Among a large number of drugs developed in the programme, N-[2-(dimethylamino)-ethyl]-acridine-4-carboxamide (DACA), first synthesised by Professor Denny, showed excellent activity against a mouse lung adenocarcinoma. It underwent clinical trial, but dose escalation was prevented by ion channel toxicity. Subsequent work led to the DACA derivative SN 28049, which had increased potency and reduced ion channel toxicity. Mode of action studies suggested that both amsacrine and DACA target the enzyme DNA topoisomerase II but with a different balance of cellular consequences. As primarily a topoisomerase II poison, amsacrine acts to turn the enzyme into a DNA-damaging agent. As primarily topoisomerase II catalytic inhibitors, DACA and SN 28049 act to inhibit the segregation of daughter chromatids during anaphase. The balance between these two actions, one cell cycle phase specific and the other nonspecific, together with pharmacokinetic, cytokinetic and immunogenic considerations, provides links between the actions of acridine derivatives and anthracyclines such as doxorubicin. They also provide insights into the action of cytotoxic DNA-binding drugs.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos , DNA de Neoplasias/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores da Topoisomerase II , Adenocarcinoma de Pulmão/história , Adenocarcinoma de Pulmão/metabolismo , Amsacrina/química , Amsacrina/história , Amsacrina/farmacocinética , Amsacrina/uso terapêutico , Anáfase/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/história , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Cromátides/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , História do Século XX , História do Século XXI , Humanos , Neoplasias Pulmonares/história , Neoplasias Pulmonares/metabolismo , Camundongos , Naftiridinas/química , Naftiridinas/farmacocinética , Naftiridinas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacocinética , Inibidores da Topoisomerase II/uso terapêutico
14.
Curr Genet ; 67(3): 447-459, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33404730

RESUMO

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Centrômero/genética , Cromátides/genética , Ácido Glutâmico/genética , Mutação/genética , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Troca de Cromátide Irmã/genética
15.
Exp Cell Res ; 399(2): 112455, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33400935

RESUMO

During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Cromátides/ultraestrutura , Pareamento Cromossômico/fisiologia , DNA/química , DNA/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Complexo Sinaptonêmico/fisiologia , Complexo Sinaptonêmico/ultraestrutura
16.
PLoS Genet ; 17(1): e1009304, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33513180

RESUMO

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.


Assuntos
Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Cinetocoros , Animais , DNA Topoisomerases Tipo II/genética , Drosophila melanogaster/genética , Larva/genética , Metáfase/genética , Microtúbulos/genética , Mitose/genética , Fuso Acromático/genética
17.
J Exp Bot ; 72(2): 254-267, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33029645

RESUMO

Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.


Assuntos
Festuca , Lolium , Cromátides/genética , Troca Genética , Festuca/genética , Lolium/genética , Meiose/genética , Cebolas
18.
EMBO J ; 40(1): e105393, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33155682

RESUMO

The juxtaposition of intracellular DNA segments, together with the DNA-passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Ciclo Celular/fisiologia , Cromátides/metabolismo , Cromatina/metabolismo , Saccharomyces cerevisiae/metabolismo
19.
Trends Biochem Sci ; 46(2): 169-170, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33342665

Assuntos
Cromátides
20.
Methods Mol Biol ; 2153: 383-393, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840793

RESUMO

The semiconservative nature of DNA replication allows the differential labeling of sister chromatids that is the fundamental requirement to perform the sister-chromatid exchange (SCE) assay. SCE assay is a powerful technique to visually detect the physical exchange of DNA between sister chromatids. SCEs could result as a consequence of DNA damage repair by homologous recombination (HR) during DNA replication. Here, we provide the detailed protocol to perform the SCE assay in cultured human cells. Cells are exposed to the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) during two cell cycles, resulting in the two sister chromatids having differential incorporation of the analog. After metaphase spreads preparation and further processing, SCEs are nicely visualized under the microscope.


Assuntos
Bromodesoxiuridina/farmacologia , Cromátides/genética , Cariotipagem/métodos , Troca de Cromátide Irmã/efeitos dos fármacos , Técnicas de Cultura de Células , Ciclo Celular , Cromátides/efeitos dos fármacos , Replicação do DNA , Células HeLa , Humanos
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