RESUMEN

The effects of drugs of abuse on oral mucosa are only partly understood. The aims of the present study were to: (1) evaluate the frequency of nuclear changes in normal-appearing oral mucosa of alcoholics and crack cocaine users and (2) assess their association with cell proliferation rate. Oral smears were obtained from the border of the tongue and floor of the mouth of 26 crack cocaine users (24 males and 2 females), 29 alcoholics (17 males and 12 females), and 35 controls (17 males and 18 females). Histological slides were submitted to Feulgen staining to assess the frequency of micronuclei (MN), binucleated cells (BN), broken eggs (BE), and karyorrhexis (KR). A significant increase in the frequency of MN was observed in cells exfoliated from the tongue of crack cocaine users (p = 0.01), and alcoholics showed a higher frequency of KR in cells obtained from the floor of the mouth (p = 0.01). Our findings suggest that the use of crack cocaine induces clastogenic effects, whereas alcoholism is associated with higher degrees of keratinization in the floor of the mouth.

Asunto(s)

Alcoholismo/patología , Núcleo Celular/patología , Trastornos Relacionados con Cocaína/patología , Cocaína Crack , Mucosa Bucal/patología , Adulto , Alcohólicos , Proliferación Celular/efectos de los fármacos , Estudios Transversales , Femenino , Humanos , Queratinas/metabolismo , Masculino , Pruebas de Micronúcleos , Persona de Mediana Edad , Boca/patología , Mutágenos/toxicidad , Salud Bucal , Lengua/patología , Adulto Joven

RESUMEN

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

Asunto(s)

Proteínas de Ciclo Celular , Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Ciclo Celular/fisiología , Quinasa de Punto de Control 2 , ADN de Hongos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidroxiurea/farmacología , Mutación , Conformación de Ácido Nucleico , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Origen de Réplica , Ribonucleótido Reductasas/antagonistas & inhibidores , Saccharomycetales

RESUMEN

Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.

Asunto(s)

Adaptación Fisiológica , Antígenos Nucleares , Daño del ADN , ADN Helicasas , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Enzimas Reparadoras del ADN , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Fase G2 , Eliminación de Gen , Cinética , Autoantígeno Ku , Mitosis , Proteínas Nucleares/genética , Saccharomyces cerevisiae/citología

RESUMEN

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.

Asunto(s)

Adaptación Biológica/genética , Antígenos Nucleares , Ciclo Celular , Daño del ADN/genética , ADN Helicasas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Anafase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Proteínas Fúngicas/metabolismo , Fase G2 , Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Cinética , Autoantígeno Ku , Mitosis , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas de Unión al ARN , Recombinación Genética , Proteína de Replicación A , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Ácido Nucleico

RESUMEN

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.

Asunto(s)

Proteína Quinasa CDC2/metabolismo , ADN Helicasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Western Blotting , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Separación Celular , Quinasa de Punto de Control 2 , Daño del ADN , ADN Helicasas/genética , Reparación del ADN , Proteínas de Unión al ADN , Citometría de Flujo , Proteínas Fúngicas/genética , Genotipo , Péptidos y Proteínas de Señalización Intracelular , Metilmetanosulfonato/farmacología , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Proteínas Nucleares , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Recombinación Genética , Fase S , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Factores de Tiempo

RESUMEN

In response to genotoxic agents and cell cycle blocks all eukaryotic cells activate a set of surveillance mechanims called checkpoints. A subset of these mechanisms is represented by the DNA damage checkpoint, which is triggered by DNA lesions. The activation of this signal transduction pathway leads to a delay of cell cycle progression to prevent replication and segregation of damaged DNA molecules, and to induce transcription of several DNA repair genes. The yeast Saccharomyces cerevisiae has been invaluable in genetically dissecting the DNA damage checkpoint pathway and recent findings have provided new insights into the architecture of checkpoint protein complexes, in their order of function and in the mechanisms controlling DNA replication in response to DNA damage.

Asunto(s)

Ciclo Celular/genética , Daño del ADN/fisiología , Replicación del ADN , Saccharomyces cerevisiae/genética , Transducción de Señal , Transcripción Genética

Asunto(s)

Cromosomas Fúngicos/genética , Daño del ADN/genética , ADN de Hongos/genética , Saccharomyces cerevisiae/genética , Ciclo Celular/fisiología , ADN de Cadena Simple/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Saccharomyces cerevisiae/citología

RESUMEN

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.

Asunto(s)

Proteínas de Ciclo Celular , Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ciclo Celular , Quinasa de Punto de Control 2 , Activación Enzimática , Proteínas Fúngicas/metabolismo , Fase G1 , Genotipo , Modelos Genéticos , Mutagénesis , Fosforilación , Fase S , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología

RESUMEN

Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.

Asunto(s)

Daño del ADN , Fase S , Schizosaccharomyces/genética , Schizosaccharomyces/citología

RESUMEN

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

Asunto(s)

Proteínas de Ciclo Celular , Daño del ADN/genética , Replicación del ADN/genética , Proteínas Serina-Treonina Quinasas , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Western Blotting , Ciclo Celular/genética , Quinasa de Punto de Control 2 , ADN/biosíntesis , ADN Primasa , Estabilidad de Enzimas/genética , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Interfase/genética , Metilmetanosulfonato/farmacología , Mitosis/genética , Modelos Biológicos , Mutagénesis Sitio-Dirigida/genética , Mutágenos/farmacología , Mutación/genética , Fosforilación , Proteínas Quinasas , ARN Nucleotidiltransferasas/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Temperatura , Rayos Ultravioleta/efectos adversos