RESUMO
The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.
Assuntos
Proteínas de Ligação a DNA , Neoplasias Ovarianas , Humanos , Feminino , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Células HeLa , Medicina de Precisão , Proteína BRCA2/metabolismo , Reparo do DNA , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Linhagem Celular TumoralRESUMO
The data presented in this manuscript presents the characterisation spectra of three hyperbranched polymers as discussed in the paper "Folic Acid and Rhodamine Labelled pH Responsive Hyperbranched Polymers: synthesis, characterisation and cell uptake studies" [1]. Characterisation of polymers was performed via 1H Nuclear Magnetic Resonance (1H NMR) and Size Exclusion Chromatography (SEC). pH responsive characteristics were observed via Dynamic Light Scattering (DLS). The data for characterisation of folate conjugated hyperbranched polymer is presented as 1H NMR, Ultra Violet Visible (UV-VIS) spectra and DLS measurements. Further data is presented detailing the experiments for the synthesis of monomers 2-propyl acrylic acid (PAA) and disulfide diacrylate (DSDA), with the full synthesis of folic acid-poly (ethylene glycol) (PEG) linker, rhodamine B ethylenediamine linker and bioconjugation reactions also detailed.
RESUMO
Epistasis mapping, in which the phenotype that emerges from combining pairs of mutations is measured quantitatively, is a powerful tool for unbiased study of gene function. When performed at a large scale, this approach has been used to assign function to previously uncharacterized genes, define functional modules and pathways, and study their cross talk. These experiments rely heavily on methods for rapid sampling of binary combinations of mutant alleles by systematic generation of a series of double mutants. Epistasis mapping technologies now exist in various model systems. Here we provide an overview of different epistasis mapping technologies, including the pombe epistasis mapper (PEM) system designed for the collection of quantitative genetic interaction data in fission yeast Schizosaccharomyces pombe Comprising a series of high-throughput selection steps for generation and characterization of double mutants, the PEM system has provided insight into a wide range of biological processes as well as facilitated evolutionary analysis of genetic interactomes across different species.
Assuntos
Mapeamento Cromossômico/métodos , Epistasia Genética , Genes Fúngicos , Genética Microbiana/métodos , Schizosaccharomyces/genética , Mutação , Seleção GenéticaRESUMO
This protocol describes computational analysis of genetic interaction screens, ranging from data capture (plate imaging) to downstream analyses. Plate imaging approaches using both digital camera and office flatbed scanners are included, along with a protocol for the extraction of colony size measurements from the resulting images. A commonly used genetic interaction scoring method, calculation of the S-score, is discussed. These methods require minimal computer skills, but some familiarity with MATLAB and Linux/Unix is a plus. Finally, an outline for using clustering and visualization software for analysis of resulting data sets is provided.
Assuntos
Bioestatística/métodos , Mapeamento Cromossômico/métodos , Epistasia Genética , Genética Microbiana/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Óptica/métodos , Schizosaccharomyces/genética , Análise por Conglomerados , Genes Fúngicos , Mutação , Schizosaccharomyces/crescimento & desenvolvimento , Seleção GenéticaRESUMO
In laboratories in which a colony-replicating robot is not available, manual replication provides a good, low-cost alternative for genetic interaction screening using the Pombe Epistasis Mapper (PEM) system. The protocol presented here describes the minimum number of steps required to identify genetic interactions. First, a query deletion is introduced to a library of deletion mutants by mating. Through a series of subsequent selection steps, single and double mutants are isolated and analyzed.
Assuntos
Mapeamento Cromossômico/instrumentação , Mapeamento Cromossômico/métodos , Epistasia Genética , Genes Fúngicos , Genética Microbiana/instrumentação , Genética Microbiana/métodos , Schizosaccharomyces/genética , Mutação , Seleção GenéticaRESUMO
In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside transporter human equilibrative nucleoside transporter 1 (hENT1) and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1-in addition to repressing dNTP synthesis-together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.
RESUMO
Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Ubiquitinação , Raios Ultravioleta , Dano ao DNA , Regiões Promotoras Genéticas , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Leveduras/enzimologia , Leveduras/genética , Leveduras/metabolismo , Leveduras/efeitos da radiaçãoRESUMO
During S-phase replication forks can stall at specific genetic loci. At some loci, the stalling events depend on the replisome components Schizosaccharomyces pombe Swi1 (Saccharomyces cerevisiae Tof1) and Swi3 (S. cerevisiae Csm3) as well as factors that bind DNA in a site-specific manner. Using a new genetic screen we identified Mrc1 (S. cerevisiae Mrc1/metazoan Claspin) as a replisome component involved in replication stalling. Mrc1 is known to form a sub-complex with Swi1 and Swi3 within the replisome and is required for the intra-S phase checkpoint activation. This discovery is surprising as several studies show that S. cerevisiae Mrc1 is not required for replication barrier activity. In contrast, we show that deletion of S. pombe mrc1 leads to an approximately three-fold reduction in barrier activity at several barriers and that Mrc1's role in replication fork stalling is independent of its role in checkpoint activation. Instead, S. pombe Mrc1 mediated fork stalling requires the presence of a functional copy of its phylogenetically conserved DNA binding domain. Interestingly, this domain is on the sequence level absent from S. cerevisiae Mrc1. Our study indicates that direct interactions between the eukaryotic replisome and the DNA are important for site-specific replication stalling.
Assuntos
DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Testes Genéticos/métodos , Fase S , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Topoisomerases can release topological stress and resolve DNA catenanes by a DNA strand breakage and re-ligation mechanism. During the lifetime of the DNA break, the topoisomerase remains covalently linked to the DNA and removes itself when the break is re-ligated. While the lifetime of a covalent topoisomerase-DNA complex is usually short, several clinically important cancer drugs kill cancer cells by inhibiting the removal of covalently linked topoisomerases. The topoisomerase-like protein Spo11 is responsible for meiotic double strand break formation. Spo11 is not able to remove itself and is removed by nucleolytic cleavage. This chapter describes a method which allows the reproducible and quantitative detection of proteins covalently bound to the DNA.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Endodesoxirribonucleases/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
DNA topoisomerases regulate the topological state of the DNA double helix and are key enzymes in the processes of DNA replication, transcription and genome stability. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome wide how DNA topoisomerases I and II affect chromatin dynamics and gene expression in vivo. We show that topoisomerase I activity is directly required for efficient nucleosome disassembly at gene promoter regions. Lack of topoisomerase activity results in increased nucleosome occupancy, perturbed histone modifications and reduced transcription from these promoters. Strong correlative evidence suggests that topoisomerase I cooperates with the ATP-dependent chromatin remodeller Hrp1 in nucleosome disassembly. Our study links topoisomerase activity to the maintenance of open chromatin and regulating transcription in vivo.
Assuntos
Cromatina/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Schizosaccharomyces/genética , Transcrição GênicaRESUMO
Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3' and 5' ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3' single-strand overhang at "clean" DSBs, thus initiating homologous recombination (HR)-dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3' single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIP(S332A/-/-) cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP(+/-/-) cells. Finally, CtIP(S332A/-/-)BRCA1(-/-) and CtIP(+/-/-)BRCA1(-/-) showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.
Assuntos
Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/metabolismo , Animais , Proteína BRCA1/genética , Ciclo Celular , Linhagem Celular Tumoral , Galinhas , DNA/genética , Proteínas Nucleares/genética , Ligação Proteica , Recombinação GenéticaRESUMO
The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Reparo do DNA , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de SequênciaRESUMO
The maintenance of genome stability is essential for an organism to avoid cell death and cancer. Based on screens for mutant sensitivity against DNA damaging agents a large number of DNA repair and DNA damage checkpoint genes have previously been identified in genetically amenable model organisms. These screens have however not been exhaustive and various genes have been, and remain to be, identified by other means. We therefore screened a genome-wide Schizosaccharomyces pombe deletion library for mutants sensitive against various DNA damaging agents. Screening the library on different concentrations of these genotoxins allowed us to assign a semi-quantitative score to each mutant expressing the degree of sensitivity. We isolated a total of 229 mutants which show sensitivity to one or more of the DNA damaging agents used. This set of mutants was significantly enriched for processes involved in DNA replication, DNA repair, DNA damage checkpoint, response to UV, mating type switching, telomere length maintenance and meiosis, and also for processes involved in the establishment and maintenance of chromatin architecture (notably members of the SAGA complex), transcription (members of the CCR4-Not complex) and microtubule related processes (members of the DASH complex). We also identified 23 sensitive mutants which had previously been classified as "sequence orphan" or as "conserved hypothetical". Among these, we identified genes showing extensive homology to CtIP, Stra13, Ybp1/Ybp2, Human Fragile X mental retardation interacting protein NUFIP1, and Aprataxin. The identification of these homologues will provide a basis for the further characterisation of the role of these conserved proteins in the genetically amenable model organism S. pombe.
Assuntos
Deleção de Genes , Genoma Fúngico , Instabilidade Genômica , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Sequência de Aminoácidos , Antineoplásicos Alquilantes/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Mutação/genética , Schizosaccharomyces/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacosRESUMO
For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32(Mre11) nuclease activity is involved in the removal of both Top2 from 5' DNA ends as well as Top1 from 3' ends in vivo. A ctp1(CtIP) deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1(CtIP) plays distinct roles in removing topoisomerases from 5' and 3' DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Camptotecina/farmacologia , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/enzimologia , Nucléolo Celular/efeitos da radiação , Etoposídeo/análogos & derivados , Etoposídeo/farmacologia , Raios gama , Metanossulfonato de Metila/farmacologia , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/efeitos da radiaçãoRESUMO
The evolutionarily conserved Mre11/Rad50/Nbs1 (MRN) complex is involved in various aspects of meiosis. Whereas available evidence suggests that the Mre11 nuclease activity might be responsible for Spo11 removal in Saccharomyces cerevisiae, this has not been confirmed experimentally. This study demonstrates for the first time that Mre11 (Schizosaccharomyces pombe Rad32(Mre11)) nuclease activity is required for the removal of Rec12(Spo11). Furthermore, we show that the CtIP homologue Ctp1 is required for Rec12(Spo11) removal, confirming functional conservation between Ctp1(CtIP) and the more distantly related Sae2 protein from Saccharomyces cerevisiae. Finally, we show that the MRN complex is required for meiotic recombination, chromatin remodeling at the ade6-M26 recombination hot spot, and formation of linear elements (which are the equivalent of the synaptonemal complex found in other eukaryotes) but that all of these functions are proficient in a rad50S mutant, which is deficient for Rec12(Spo11) removal. These observations suggest that the conserved role of the MRN complex in these meiotic functions is independent of Rec12(Spo11) removal.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Meiose , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , Endodesoxirribonucleases , Esterases/metabolismo , Deleção de Genes , Genes Dominantes , Nucleossomos/metabolismo , Fenótipo , Recombinação Genética , Esporos Fúngicos/citologia , Esporos Fúngicos/metabolismo , TemperaturaRESUMO
Tyrosyl DNA phosphodiesterase-1 (TDP1) is the gene product mutated in spinocerebellar ataxia with axonal neuropathy1 (SCAN1). SCAN1 is a hereditary ataxia that lacks extra-neurological phenotype, pointing to a critical role for TDP1 in the nervous system. Recently, we showed that TDP1 is associated with the DNA single-strand break (SSBR) repair machinery through an interaction with DNA ligase 3alpha (Lig3alpha) and that SCAN1 cells are defective in the repair of chromosomal DNA single-strand breaks (SSBs) arising from abortive Topoisomerase 1 (Top1)-DNA intermediates. Here we demonstrate that TDP1 is also required for the repair of SSBs induced by ionizing radiation (IR), though not measurably for IR-induced DNA double-strand breaks (DSBs). In addition, we provide evidence that abortive Top1 cleavage complexes are processed by the proteasome prior to the action of TDP1 in vivo, and we exploit this observation to show that the SSBR defect in SCAN1 following IR reflects, in part at least, the presence of IR-induced protein-DNA cross-links. Finally we show that TDP1 activity at abortive Top1-SSBs is stimulated by XRCC1/Lig3alpha in vitro. These data expand the type of SSBs processed by TDP1 to include those induced by ionizing radiation, and raise the possibility that TDP1 inhibitors may improve radiotherapy.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/efeitos da radiação , Diester Fosfórico Hidrolases/fisiologia , Radiação Ionizante , Sequência de Bases , Primers do DNA , Humanos , Proteínas Recombinantes/metabolismoRESUMO
The study of double-strand break repair and homologous recombination in Saccharomyces cerevisiae meiosis has provided important information about the mechanisms involved. However, it has become clear that the resulting recombination models are only partially applicable to repair in mitotic cells, where crossover formation is suppressed. In recent years our understanding of double-strand break repair and homologous recombination in Schizosaccharomyces pombe has increased significantly, and the identification of novel pathways and genes with homologues in higher eukaryotes has increased its value as a model organism for double-strand break repair. In this review we will focus on the involvement of homologous recombination and repair in different aspects of genome stability in Sz. pombe meiosis, replication and telomere maintenance. We will also discuss anti-recombination pathways (that suppress crossover formation), non-homologous end-joining, single-strand annealing and factors that influence the choice and prevalence of the different repair pathways in Sz. pombe.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Genética/genética , Schizosaccharomyces/genética , Genoma Fúngico , Mitose/genéticaRESUMO
The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4+ gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5' end to 6% at the 3' end was detected. A novel phenomenon also was discovered: a mating-type-related bias of conversion. The allele entering with the h+ parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5' end, and more often than not covers the entire gene. Restoration repair of markers at the 5' end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations.
Assuntos
Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Meiose , Recombinação Genética , Schizosaccharomyces/genética , Alelos , Centrômero/genética , Mapeamento Cromossômico , Cromossomos Fúngicos , Troca Genética , Conversão Gênica , Teste de Complementação Genética , Marcadores Genéticos , Mutação , Ácidos Nucleicos Heteroduplexes/genética , Esporos Fúngicos/genéticaRESUMO
Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner.
Assuntos
Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Meiose/genética , Recombinação Genética/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Fator 1 Ativador da Transcrição , Fatores Ativadores da Transcrição , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Genes Fúngicos/genética , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
To facilitate the N-terminal tagging of essential genes at their genomic locus and under control of their own promoters we have developed a series of novel polymerase chain reaction templates. Initially, a 1.8 kb DNA fragment is integrated upstream of the ATG of the gene of interest. This fragment encodes the tag, a loxP site, a selectable marker, an exogenous nmt1 promoter and a second loxP site. In a single homologous integration event, the gene of interest is placed under control of the thiamine regulated nmt1 promoter, allowing identification of potential integrants on the basis of phenotype. Subsequently, this integrant strain is transformed with a plasmid expressing the Cre recombinase. This results in excision of the marker and nmt1 promoter and leaves sequences encoding an in-frame tag at the N-terminus of the gene of interest under the control of its native promoter. We have created TAP-cdc22, TAP-suc22 and TAP-rad50 strains using this N-tagging system, and developed a range of vectors for introducing TAP-, (His)10HA-, (His)6Myc- and EGFP.