RESUMO
Epithelial cells undergo tubulogenesis in response to morphogens such as hepatocyte growth factor (HGF). To organize into tubules, cells must execute a complex series of morphogenetic events; however, the mechanisms that underlie the timing and sequence of these events are poorly understood. Here, we show that downstream effectors of HGF coordinately regulate successive stages of tubulogenesis. Activation of extracellular-regulated kinase (ERK) is necessary and sufficient for the initial stage, during which cells depolarize and migrate. ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. Conversely, the activity of matrix metalloproteases (MMPs) is essential for the late stage but not the initial stage. Thus, ERK and MMPs define two regulatory subprograms that act in sequence. By inducing these reciprocal signals, HGF directs the morphogenetic progression of tubule development.
Assuntos
Células Epiteliais/enzimologia , Fator de Crescimento de Hepatócito/metabolismo , Metaloproteinases da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Organogênese/fisiologia , Vísceras/embriologia , Vísceras/enzimologia , Animais , Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Cães , Células Epiteliais/citologia , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Vísceras/citologiaRESUMO
The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.
Assuntos
Proteínas de Ciclo Celular/genética , DNA Helicases/genética , Reparo do DNA , Chaperonas Moleculares/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Chaperonas Moleculares/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The chromatin-assembly factor I (CAF-I) and the replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and play a role in the maintenance of genome stability. Here, we have investigated their role in checkpoints and S-phase progression. FACS analysis of mutants lacking Asf1 or Cac1 as well as various checkpoint proteins indicated that normal rates of S-phase progression in asf1 mutants have a strong requirement for replication checkpoint proteins, whereas normal S-phase progression in cac1 mutants has only a weak requirement for either replication or DNA-damage checkpoint proteins. Furthermore, asf1 mutants had high levels of Ddc2.GFP foci that were further increased in asf1 dun1 double mutants consistent with a requirement for checkpoint proteins in S-phase progression in asf1 mutants, whereas cac1 mutants had much lower levels of Ddc2.GFP foci that were not increased by a dun1 mutation. Our data suggest that RCAF defects lead to unstable replication forks that are then stabilized by replication checkpoint proteins, whereas CAF-I defects likely cause different types of DNA damage.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Fator 1 de Modelagem da Cromatina , Dano ao DNA , Replicação do DNA , Genes Fúngicos , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Modelos Biológicos , Chaperonas Moleculares , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.