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1.
Nat Commun ; 8: 16111, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28706291

RESUMO

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a Kd of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.


Assuntos
Anticorpos/química , Proteínas ras/antagonistas & inibidores , Repetição de Anquirina , Anticorpos/imunologia , Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Células HCT116 , Células HEK293 , Humanos , Estrutura Molecular , Terapia de Alvo Molecular , Proteínas ras/imunologia
2.
Oncotarget ; 7(42): 68278-68291, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27626702

RESUMO

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.


Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Células HEK293 , Humanos , Células Jurkat , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Fenótipo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos
3.
Mol Cancer ; 14: 147, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26227951

RESUMO

BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Neoplasias , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Cisplatino/farmacologia , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Biblioteca de Peptídeos , Fenótipo , Anticorpos de Cadeia Única/farmacologia , Esferoides Celulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Trends Biotechnol ; 33(3): 163-71, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25595556

RESUMO

Owing to the challenges of cell entry, protein-based therapies have so far been restricted to extracellular targets, whereas intracellular targets have been almost exclusively addressed by small molecules. The specificity and potency of proteins would enable them to be effective intracellular drugs, provided that the proteins are delivered efficiently to appropriate intracellular compartments within specific cell types. By mimicking the natural mechanisms of toxins and other natural proteins, new intracellular delivery systems are being developed, the first of which are showing clinical efficacy. This review highlights a range of ingenious approaches designed to adapt natural entry mechanisms to facilitate delivery of proteins and open up a range of validated intracellular targets to modulation by potent and specific therapeutic drugs.


Assuntos
Terapia Biológica/métodos , Engenharia de Proteínas/métodos , Proteínas/farmacocinética , Proteínas/uso terapêutico , Humanos , Transporte Proteico , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico
5.
Mol Cancer ; 12: 11, 2013 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-23406016

RESUMO

BACKGROUND: The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a 'triple negative' breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. METHODS: The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other 'triple negative' breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. RESULTS: All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other 'triple negative' breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model. CONCLUSIONS: This study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.


Assuntos
5'-Nucleotidase/metabolismo , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/metabolismo , Proteoma/metabolismo , Anticorpos de Cadeia Única/metabolismo , 5'-Nucleotidase/imunologia , Animais , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Portadores de Fármacos/farmacologia , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Hibridomas , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Biblioteca de Peptídeos , Fenótipo , Ligação Proteica , Receptor ErbB-2 , Receptores de Estrogênio , Receptores de Progesterona , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Exp Med ; 209(7): 1273-87, 2012 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-22734046

RESUMO

Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.


Assuntos
Anticorpos Monoclonais/imunologia , Polissacarídeos Bacterianos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Linhagem Celular Tumoral , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/imunologia , Mutação , Biblioteca de Peptídeos , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Pneumonia/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/genética , Sorotipagem , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia
7.
J Biomol Screen ; 17(7): 993-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22522649

RESUMO

5'-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples.


Assuntos
5'-Nucleotidase/análise , Monofosfato de Adenosina/metabolismo , Adenosina/biossíntese , Ensaios de Triagem em Larga Escala/métodos , Nucleotidases/análise , Cromatografia Líquida de Alta Pressão/métodos , Proteínas Ligadas por GPI/análise , Humanos
8.
Cell Cycle ; 8(3): 443-53, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19177002

RESUMO

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma.


Assuntos
Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/farmacologia , Glioma/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Cromonas/farmacologia , Cromonas/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Furanos/farmacologia , Furanos/uso terapêutico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Camundongos , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico
9.
Cancer Res ; 67(12): 5840-50, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17575152

RESUMO

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.


Assuntos
Antineoplásicos/farmacologia , Furanos/farmacologia , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
DNA Repair (Amst) ; 5(7): 840-51, 2006 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-16765654

RESUMO

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.


Assuntos
DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Sequência de Bases , Ciclo Celular , Reparo do DNA , DNA Fúngico/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Genes Fúngicos , Modelos Biológicos , Complexos Multiproteicos , Mutação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Telômero/genética
12.
Cell ; 124(6): 1155-68, 2006 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-16564010

RESUMO

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex -- the KEOPS complex -- containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 cgi121 mutants. Deletion of BUD32 or other KEOPS components leads to short telomeres and a failure to add telomeres de novo to DNA double-strand breaks. Our results therefore indicate that the KEOPS complex promotes both telomere uncapping and telomere elongation.


Assuntos
Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Biblioteca Genômica , Proteínas de Saccharomyces cerevisiae/fisiologia , Telômero/fisiologia , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
13.
Genetics ; 168(1): 103-15, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15454530

RESUMO

Cell cycle arrest in response to DNA damage depends upon coordinated interactions between DNA repair and checkpoint pathways. Here we examine the role of DNA repair and checkpoint genes in responding to unprotected telomeres in budding yeast cdc13-1 mutants. We show that Exo1 is unique among the repair genes tested because like Rad9 and Rad24 checkpoint proteins, Exo1 inhibits the growth of cdc13-1 mutants at the semipermissive temperatures. In contrast Mre11, Rad50, Xrs2, and Rad27 contribute to the vitality of cdc13-1 strains grown at permissive temperatures, while Din7, Msh2, Nuc1, Rad2, Rad52, and Yen1 show no effect. Exo1 is not required for cell cycle arrest of cdc13-1 mutants at 36 degrees but is required to maintain arrest. Exo1 affects but is not essential for the production of ssDNA in subtelomeric Y' repeats of cdc13-1 mutants. However, Exo1 is critical for generating ssDNA in subtelomeric X repeats and internal single-copy sequences. Surprisingly, and in contrast to Rad24, Exo1 is not essential to generate ssDNA in X or single-copy sequences in cdc13-1 rad9Delta mutants. We conclude that Rad24 and Exo1 regulate nucleases with different properties at uncapped telomeres and propose a model to explain our findings.


Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA , DNA de Cadeia Simples/genética , Exodesoxirribonucleases/genética , Genes cdc , Saccharomyces cerevisiae/genética , Telômero/genética , Ciclo Celular/genética , Contagem de Colônia Microbiana , Primers do DNA , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Genéticos , Mutação/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Temperatura
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