RESUMEN
Environmental temperature acclimation is essential to animal survival, yet thermoregulation mechanisms remain poorly understood. We demonstrate cold tolerance in Caenorhabditis elegans as regulated by paired ADL chemosensory neurons via Ca2+-dependent endoribonuclease (EndoU) ENDU-2. Loss of ENDU-2 function results in life span, brood size, and synaptic remodeling abnormalities in addition to enhanced cold tolerance. Enzymatic ENDU-2 defects localized in the ADL and certain muscle cells led to increased cold tolerance in endu-2 mutants. Ca2+ imaging revealed ADL neurons were responsive to temperature stimuli through transient receptor potential (TRP) channels, concluding that ADL function requires ENDU-2 action in both cell-autonomous and cell-nonautonomous mechanisms. ENDU-2 is involved in caspase expression, which is central to cold tolerance and synaptic remodeling in dorsal nerve cord. We therefore conclude that ENDU-2 regulates cell type-dependent, cell-autonomous, and cell-nonautonomous cold tolerance.
Asunto(s)
Aclimatación/fisiología , Caenorhabditis elegans/enzimología , Endorribonucleasas/metabolismo , Carácter Cuantitativo Heredable , Transducción de Señal/fisiología , Sinapsis/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biosíntesis , Proteínas de Caenorhabditis elegans/genética , Caspasas/biosíntesis , Caspasas/genética , Endorribonucleasas/genética , Perfilación de la Expresión Génica , Sinapsis/genética , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1Δ mus81Δ synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1Δ rnh202Δ synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Epistasis Genética , Endonucleasas de ADN Solapado/genética , RecQ Helicasas/genética , Recombinación Genética , Ribonucleasa H/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Daño del ADN , Genes Bacterianos , Proteína Recombinante y Reparadora de ADN Rad52/genéticaRESUMEN
Genetic studies in budding yeast have previously implicated SLX5 and SLX8 in the control of genome stability and sumoylation. These genes encode RING-finger domain proteins that form a complex of unknown function. Because RING-finger proteins comprise a large class of ubiquitin (Ub) ligases, Slx5 and Slx8 were tested for this activity. Here we show that the Slx5-Slx8 complex, but not its individual subunits, stimulates several human and yeast Ub conjugating enzymes, including Ubc1, 4, 5, and Ubc13-Mms2. The RING-finger domains of both subunits are genetically required for suppression of slx sgs1Delta synthetic-lethality, and point mutations that abolish Ub ligase activity in vitro also eliminate in vivo complementation. Targets of the in vitro ubiquitination reaction include the Slx5 and Slx8 subunits themselves, and the homologous recombination proteins Rad52 and Rad57. We propose that the Slx5-Slx8 complex functions as a two-component Ub ligase in vivo and that it controls genome stability and sumoylation via ubiquitination.
Asunto(s)
ADN de Hongos/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Dedos de Zinc/fisiología , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Humanos , Dominios RING Finger/genética , Dominios RING Finger/fisiología , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Ubiquitina-Proteína LigasasRESUMEN
The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2micro circle from the mutants, suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Delta and slx8Delta mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5 and Slx8 complex is capable of interacting directly with the SUMO pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/genética , Dominios RING Finger , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Tiempo , Ubiquitina-Proteína LigasasRESUMEN
Previous studies in yeast have suggested that the SGS1 DNA helicase or the Mus81-Mms4 structure-specific endonuclease is required to suppress the accumulation of lethal recombination intermediates during DNA replication. However, the structure of these intermediates and their mechanism of the suppression are unknown. To examine this reaction, we have isolated and characterized a temperature-sensitive (ts) allele of MUS81. At the non-permissive temperature, sgs1Deltamus81(ts) cells arrest at G(2)/M phase after going through S-phase. Bulk DNA replication appears complete but is defective since the Rad53 checkpoint kinase is strongly phosphorylated under these conditions. In addition, the induction of Rad53 hyper-phosphorylation by MMS was deficient at permissive temperature. Analysis of rDNA replication intermediates at the non-permissive temperature revealed elevated pausing of replication forks at the RFB in the sgs1Deltamus81(ts) mutant and a novel linear structure that was dependent on RAD52. Pulsed-field gel electrophoresis of the mus81Delta mutant revealed an expansion of the rDNA locus depending on RAD52, in addition to fragmentation of Chr XII in the sgs1Deltamus81(ts) mutant at permissive temperature. This is the first evidence that Mus81 functions in quality control of replication forks and that it is involved in the maintenance of rDNA repeats in vivo.