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2.
DNA Res ; 28(6)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34609504

RESUMO

The complete sequencing of human centromeres, which are filled with highly repetitive elements, has long been challenging. In human centromeres, α-satellite monomers of about 171 bp in length are the basic repeating units, but α-satellite monomers constitute the higher-order repeat (HOR) units, and thousands of copies of highly homologous HOR units form large arrays, which have hampered sequence assembly of human centromeres. Because most HOR unit occurrences are covered by long reads of about 10 kb, the recent availability of much longer reads is expected to enable observation of individual HOR occurrences in terms of their single-nucleotide or structural variants. The time has come to examine the complete sequence of human centromeres.


Assuntos
Centrômero , DNA Satélite , Centrômero/genética , Humanos , Sequências Repetitivas de Ácido Nucleico
3.
Int J Mol Sci ; 22(17)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34502420

RESUMO

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum-in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues-incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.


Assuntos
Cloreto de Alumínio , Instabilidade Cromossômica/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Alumínio/farmacocinética , Alumínio/toxicidade , Cloreto de Alumínio/farmacocinética , Cloreto de Alumínio/toxicidade , Animais , Linhagem Celular , Centrômero/metabolismo , Cricetulus
4.
J Biol Chem ; 297(4): 101213, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547289

RESUMO

The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.


Assuntos
Proteína B de Centrômero/metabolismo , Centrômero/metabolismo , RNA não Traduzido/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Genética , Animais , Centrômero/genética , Proteína B de Centrômero/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA não Traduzido/genética , Fatores de Transcrição/genética
5.
PLoS One ; 16(9): e0258028, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34591908

RESUMO

Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.


Assuntos
Centrômero , Cromossomos de Plantas , DNA Ribossômico , Histonas/genética , Lycoris/genética , Imunoprecipitação da Cromatina , Amplificação de Genes , Deleção de Genes , Histonas/metabolismo , Hibridização in Situ Fluorescente , Cariótipo , Lycoris/metabolismo
6.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342353

RESUMO

Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although mis-expression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. This article has an associated First Person interview with the first author of the paper.


Assuntos
Centrômero , Cinetocoros , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Centrômero/metabolismo , Segregação de Cromossomos , Humanos , Cinetocoros/metabolismo , Mitose , Fosforilação , Transcrição Genética
7.
Cells ; 10(7)2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34359988

RESUMO

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.


Assuntos
Núcleo Celular/genética , Cromossomos de Mamíferos/genética , Murinae/genética , Animais , Células da Medula Óssea/metabolismo , Centrômero/genética , Coloração Cromossômica , DNA/genética , Fibroblastos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Interfase , Metáfase/genética
8.
Prog Mol Subcell Biol ; 60: 203-234, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386877

RESUMO

Here we present three interesting novel human Higher-Order Repeats (HORs) discovered using the HOR-searching method with GRM algorithm: (a) The novel Neuroblastoma Breakpoint Family gene (NBPF) 3mer HOR, discovered applying GRM algorithm to human chromosome 1 (Paar et al., Mol Biol Evol 28:1877-1892, 2011). NBPF 3mer HOR is based on previously known ~1.6 kb NBPF primary repeat monomers (known as DUF1220 domain) in human chromosome 1, but the NBPF HOR was not known before its discovery by using GRM. It should be stressed that the NBPF HOR presents a unique human-specific pattern, distinguishing human from nonhuman primates. (b) The novel quartic HOR (2mer⊃2mer⊃9mer) discovered using the GRM algorithm for analysis of hornerin genes in human chromosome 1 (Paar et al., Mol Biol Evol 28:1877-1892, 2011). This quartic HOR is based on 39 bp hornerin primary repeat monomer in human chromosome 1. To our knowledge, this is the first known case of quartic HOR, with four levels of hierarchy of HOR organization. (c) The novel 33mer alpha satellite HOR in human chromosome 21, discovered using the GRM algorithm (Gluncic et al., Sci Rep 9:12629, 2019). This 33mer HOR in the smallest human chromosome is the largest alpha satellite HOR copy among all 22 somatic human chromosomes. Moreover, the same 33mer HOR is present in the hg38 human genome assembly of four human chromosomes: 21, 22, 13, and 14. We point out that the DUF1220 encoding genomic structures in NBPF genes in human chromosome 1, recently studied and related to the brain evolution and pathologies and cognitive aptitude, can be considered in the framework of the general concept of HORs, already extensively studied in genomics, especially in the centromeric region.


Assuntos
Cromossomos Humanos Par 21 , Neuroblastoma , Animais , Centrômero , DNA Satélite , Éxons , Genoma Humano/genética , Humanos , Neuroblastoma/genética
9.
Prog Mol Subcell Biol ; 60: 169-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386876

RESUMO

Centromeres are chromosomal regions that are essential for the faithful transmission of genetic material through each cell division. They represent the chromosomal platform on which assembles a protein complex, the kinetochore, which mediates attachment to the mitotic spindle. In most organisms, centromeres assemble on large arrays of tandem satellite repeats, although their DNA sequences and organization are highly divergent among species. It has become evident that centromeres are not defined by underlying DNA sequences, but are instead epigenetically defined by the deposition of the centromere-specific histone H3 variant, CENP-A. In addition, and although long regarded as silent chromosomal loci, centromeres are in fact transcriptionally competent in most species, yet at low levels in normal somatic cells, but where the resulting transcripts participate in centromere architecture, identity, and function. In this chapter, we discuss the various roles proposed for centromere transcription and their transcripts, and the potential molecular mechanisms involved. We also discuss pathological cases in which unscheduled transcription of centromeric repeats or aberrant accumulation of their transcripts are pathological signatures of chromosomal instability diseases. In sum, tight regulation of centromeric satellite repeats transcription is critical for healthy development and tissue homeostasis, and thus prevents the emergence of disease states.


Assuntos
Centrômero , Cromatina , Centrômero/genética , Proteína Centromérica A/genética , Cinetocoros , Transcrição Genética/genética
10.
mBio ; 12(4): e0147621, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34340554

RESUMO

2009 saw the first description of Candida auris, a yeast pathogen of humans. C. auris has since grown into a global problem in intensive care settings, where it causes systemic infections in patients with underlying health issues. Recent whole-genome sequencing has discerned five C. auris clades with distinct phenotypic features which display genomic divergence on a DNA sequence and a chromosome structure level. In the absence of sexual reproduction in C. auris, the mechanism(s) behind the rapid genomic evolution of this emerging killer yeast has remained obscure. Yet, one important bit of information about chromosome organization was missing, the identification of the centromeres. In a recent study, Sanyal and coworkers (A. Narayanan, R. N. Vadnala, P. Ganguly, P. Selvakumar, et al., mBio 12:e00905-21, 2021, https://doi.org/10.1128/mBio.00905-21) filled this knowledge gap by mapping the centromeres in C. auris and its close relatives. This represents a major advance in the chromosome biology of the Candida/Clavispora clade.


Assuntos
Candidíase , Saccharomycetales , Sequência de Bases , Candida/genética , Centrômero , Humanos
11.
Appl Microbiol Biotechnol ; 105(14-15): 5959-5972, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34357429

RESUMO

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.


Assuntos
Origem de Replicação , Yarrowia , Centrômero , Replicação do DNA , Engenharia Metabólica , Plasmídeos/genética , Yarrowia/genética
12.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445507

RESUMO

Epigenetic regulators play a crucial role in establishing and maintaining gene expression states. To date, the main efforts to study cellular heterogeneity have focused on elucidating the variable nature of the chromatin landscape. Specific chromatin organisation is fundamental for normal organogenesis and developmental homeostasis and can be affected by different environmental factors. The latter can lead to detrimental alterations in gene transcription, as well as pathological conditions such as cancer. Epigenetic marks regulate the transcriptional output of cells. Centromeres are chromosome structures that are epigenetically regulated and are crucial for accurate segregation. The advent of single-cell epigenetic profiling has provided finer analytical resolution, exposing the intrinsic peculiarities of different cells within an apparently homogenous population. In this review, we discuss recent advances in methodologies applied to epigenetics, such as CUT&RUN and CUT&TAG. Then, we compare standard and emerging single-cell techniques and their relevance for investigating human diseases. Finally, we describe emerging methodologies that investigate centromeric chromatin specification and neocentromere formation.


Assuntos
Centrômero/metabolismo , Cromatina/metabolismo , Análise de Célula Única/métodos , Montagem e Desmontagem da Cromatina , Epigênese Genética , Humanos
13.
Nucleic Acids Res ; 49(16): 9174-9193, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417622

RESUMO

To investigate how exogenous DNA concatemerizes to form episomal artificial chromosomes (ACs), acquire equal segregation ability and maintain stable holocentromeres, we injected DNA sequences with different features, including sequences that are repetitive or complex, and sequences with different AT-contents, into the gonad of Caenorhabditis elegans to form ACs in embryos, and monitored AC mitotic segregation. We demonstrated that AT-poor sequences (26% AT-content) delayed the acquisition of segregation competency of newly formed ACs. We also co-injected fragmented Saccharomyces cerevisiae genomic DNA, differentially expressed fluorescent markers and ubiquitously expressed selectable marker to construct a less repetitive, more complex AC. We sequenced the whole genome of a strain which propagates this AC through multiple generations, and de novo assembled the AC sequences. We discovered CENP-AHCP-3 domains/peaks are distributed along the AC, as in endogenous chromosomes, suggesting a holocentric architecture. We found that CENP-AHCP-3 binds to the unexpressed marker genes and many fragmented yeast sequences, but is excluded in the yeast extremely high-AT-content centromeric and mitochondrial DNA (> 83% AT-content) on the AC. We identified A-rich motifs in CENP-AHCP-3 domains/peaks on the AC and on endogenous chromosomes, which have some similarity with each other and similarity to some non-germline transcription factor binding sites.


Assuntos
Segregação de Cromossomos , Cromossomos Artificiais/genética , Mitose , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Centrômero/genética , Centrômero/metabolismo , Sequência Rica em GC , Proteínas de Choque Térmico/metabolismo , Ligação Proteica , Saccharomyces cerevisiae
14.
Science ; 373(6555): 655-662, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353948

RESUMO

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.


Assuntos
Genoma de Planta , Anotação de Sequência Molecular , Zea mays/genética , Centrômero/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Metilação de DNA , Resistência à Doença/genética , Genes de Plantas , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Herança Multifatorial/genética , Fenótipo , Doenças das Plantas , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Tetraploidia , Transcriptoma , Sequenciamento Completo do Genoma
15.
Theor Appl Genet ; 134(11): 3675-3686, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34368889

RESUMO

KEY MESSAGE: An Oligo-FISH barcode system was developed for two model legumes, allowing the identification of all cowpea and common bean chromosomes in a single FISH experiment, and revealing new chromosome rearrangements. The FISH barcode system emerges as an effective tool to understand the chromosome evolution of economically important legumes and their related species. Current status on plant cytogenetic and cytogenomic research has allowed the selection and design of oligo-specific probes to individually identify each chromosome of the karyotype in a target species. Here, we developed the first chromosome identification system for legumes based on oligo-FISH barcode probes. We selected conserved genomic regions between Vigna unguiculata (Vu, cowpea) and Phaseolus vulgaris (Pv, common bean) (diverged ~ 9.7-15 Mya), using cowpea as a reference, to produce a unique barcode pattern for each species. We combined our oligo-FISH barcode pattern with a set of previously developed FISH probes based on BACs and ribosomal DNA sequences. In addition, we integrated our FISH maps with genome sequence data. Based on this integrated analysis, we confirmed two translocation events (involving chromosomes 1, 5, and 8; and chromosomes 2 and 3) between both species. The application of the oligo-based probes allowed us to demonstrate the participation of chromosome 5 in the translocation complex for the first time. Additionally, we detailed a pericentric inversion on chromosome 4 and identified a new paracentric inversion on chromosome 10. We also detected centromere repositioning associated with chromosomes 2, 3, 5, 7, and 9, confirming previous results for chromosomes 2 and 3. This first barcode system for legumes can be applied for karyotyping other Phaseolinae species, especially non-model, orphan crop species lacking genomic assemblies and cytogenetic maps, expanding our understanding of the chromosome evolution and genome organization of this economically important legume group.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Hibridização in Situ Fluorescente , Cariotipagem/métodos , Phaseolus/genética , Vigna/genética , Centrômero , Cromossomos de Plantas/genética , Sondas Moleculares
16.
Nucleic Acids Res ; 49(16): 9053-9065, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34352103

RESUMO

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.


Assuntos
Ciclo Celular , Centrômero/metabolismo , Nucleossomos/química , Animais , Caenorhabditis elegans , Centrômero/química , Montagem e Desmontagem da Cromatina , Simulação por Computador , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo
17.
Elife ; 102021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34286695

RESUMO

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Citidina Trifosfato/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Centrômero/metabolismo , Cromossomos Bacterianos , Citidina Trifosfato/genética , DNA Primase , DNA Bacteriano , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Plasmídeos , Ligação Proteica , Pirofosfatases
18.
Elife ; 102021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34250901

RESUMO

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single-molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific DNA. This requires the presence of a parS loading site and is prevented by protein roadblocks, consistent with one-dimensional diffusion by a sliding clamp. ParB diffusion on non-specific DNA is corroborated by direct visualization and quantification of movement of individual quantum dot labelled ParB. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations.


Assuntos
Proteínas de Bactérias/metabolismo , Citidina Trifosfato/metabolismo , DNA Bacteriano/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Centrômero/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Hidrólise , Ligação Proteica , Pirofosfatases/metabolismo
19.
Elife ; 102021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34308839

RESUMO

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Divisão Celular , Centrômero/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/química , Proteínas F-Box/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Organismos Geneticamente Modificados , Fosforilação , Estabilidade Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Ubiquitina-Proteína Ligases/genética
20.
Cytogenet Genome Res ; 161(5): 272-277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34289478

RESUMO

The genus Dracaena is the main source of dragon's blood, which is a plant resin and has been used as traditional medicine since ancient times in different civilizations. However, the chromosome numbers and karyotypes present in this genus remain poorly understood. In this study, fluorescence in situ hybridization (FISH) using oligonucleotide probes for ribosomal DNAs (5S and 45S rDNA) and telomeric repeats (TTTAGGG)3 was applied to analyze 4 related species: Dracaena terniflora Roxb., Dracaena cambodiana Pierre ex Gagnep., Aizong (Dracaena sp.), and Dracaena cochinchinensis (Lour.) S.C. Chen. In all 4 species, both 5S and 45S rDNA showed hybridization signals in the paracentromeric region of a pair of chromosomes; the sizes of the 45S rDNA signals were larger than those of the 5S rDNA. Importantly, the telomeric repeat signals were located in the telomeric regions of almost all chromosomes. The results indicated that the chromosome number of all 4 Dracaena species is 2n = 40, and the lengths of the mitotic metaphase chromosomes range from 0.99 to 2.98 µm. Our results provide useful cytogenetic information, which will be beneficial to future studies in genome structure of the genus Dracaena.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/química , Dracaena/genética , Cariótipo , Centrômero , China , Dracaena/classificação , Hibridização in Situ Fluorescente/métodos , Cariotipagem/métodos , Filogeografia , RNA Ribossômico/genética , RNA Ribossômico 5S/genética , Telômero
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