RESUMO
The Sgs1 protein from Saccharomyces cerevisiae is a member of the RecQ helicases. Defects in RecQ helicases result in premature aging phenotypes in both yeasts and humans, which appear to be promoted by replicative stress. Yeast rad27 mutants also suffer from premature aging. As the human Rad27p and Sgs1p homologs interact, a similar interaction between the yeast proteins could be important for promoting longevity in S. cerevisiae. We tested the contribution of a potential interaction between Rad27p and Sgs1p to longevity by analyzing lifespan and parameters associated with longevity in rad27 and sgs1 mutants. The carbon source supporting growth also modulated longevity as evaluated by replicative and chronological lifespan measurements. Growth on glycerol promoted chronological lifespan, while maximum replicative lifespan was obtained with glucose-supported growth. In comparison to the individual mutants, the sgs1 rad27 double mutant displayed a shortened replicative lifespan and was also more sensitive to DNA-damaging agents. In addition to promoting replicative lifespan, the activity of Rad27p was critical for achieving full chronological lifespan. The rad27 mutants exhibited increased oxidative stress levels along with an elevated spontaneous mutation rate. Removal of Sgs1p activity additionally increased the oxidative stress and spontaneous mutation rate in rad27 mutants without affecting the chronological lifespan.
Assuntos
Ciclo Celular/fisiologia , Endonucleases Flap/genética , Glucose/metabolismo , Glicerol/metabolismo , Mutação/genética , RecQ Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Humanos , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence resonance energy transfer (FRET) and immune-precipitation experiments to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. We verify the WRN-RAD51 and WRN-RAD54B direct interaction in vitro. Our data support a role for WRN also in the recombination step of ICL repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA , Ensaio de Imunoadsorção Enzimática , Exodesoxirribonucleases , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/efeitos dos fármacos , Rad51 Recombinase/genética , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.
