RESUMO
The Swi4-Swi6 family of transcription factors confers G1/S specific transcription in budding and fission yeast. These proteins contain four ankyrin repeats, which are present in a large number of functionally diverse proteins and have been shown to be important for protein-protein interaction. However, no specific sequence has been identified that is diagnostic of an ankyrin repeat-interacting protein. To determine the function of the ankyrin repeats of Swi6, we generated both random and site-directed mutations within the ankyrin repeat domain of Swi6 and assayed the transcriptional function of these mutant swi6 alleles. We found six single mutations, scattered within the first and the fourth repeats, that generate a temperature-sensitive Swi6 protein. In addition, we found that alanine substitutions for the most conserved residues in each repeat were highly deleterious and also confer temperature sensitivity. Most of these swi6 alleles are able to form ternary complexes with Swi4 and DNA, but these complexes display reduced mobility in band-shift gels, suggesting a dramatic conformational change. We have modeled the ankyrin repeats of Swi6 using the coordinates derived for 53BP2 and find that, despite its low level of sequence conservation, these modeling studies and our mutation data are consistent with Swi6 having a structure very similar to that of 53BP2. Moreover, all but one of our single mutants and all of the site-directed mutants disrupt critical structural features of the predicted folding pattern of these repeats. We conclude that the ankyrin repeats play a major structural role in Swi6. Ankyrin repeats are unlikely to have inherent protein or DNA binding properties. However, they form a characteristic and stable structure with surfaces that may be tailored for many different macromolecular interactions.
Assuntos
Repetição de Anquirina/genética , Proteínas Fúngicas/genética , Mutação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/química , Immunoblotting , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Conformação Proteica , Temperatura , Fatores de Transcrição/química , Transcrição GênicaRESUMO
Budding yeast possesses a checkpoint-dependent mechanism of delaying G1 progression in response to UV and ionizing radiation DNA damage. We have shown that after a pulse of DNA damage in G1 with the alkylating agent MMS, there is also a MEC1-, RAD53-, and RAD9-dependent delay in G1. This delay occurs at or before Start, as the MMS-treated cells do not bud, remain sensitive to alpha-factor, and have low CLN1 and CLN2 transcript levels for a longer time than untreated cells. We further show that MMS directly and reversibly down-regulates CLN1 and CLN2 transcript levels. The initial drop in CLN transcript levels in MMS is not RAD53 dependent, but the kinetics of reaccumulation of CLN messages as cells recover from the damage is faster in rad53-11 cells than in wild type cells. This is not an indirect effect of faster progression through G1, because CLN transcripts reaccumulate faster in rad53-11 mutants arrested in G1 as well. In addition, the recovery of CLN mRNA levels can be also hastened by a SWI6 deletion or by overexpression of the truncated Swi4 (Swi4-t) that lacks the carboxy-terminal domain through which Swi4 associates with Swi6. This indicates that both Rad53 and Swi6 are negative regulators of CLN expression after DNA damage. Finally, Swi6 undergoes an MMS-inducible, RAD53-dependent phosphorylation in G1 cells, and Rad53, immunoprecipitated from MMS-treated cells, phosphorylates Swi6 in vitro. On the basis of these observations, we suggest that the Rad53-dependent phosphorylation of Swi6 may delay the transition to S phase by inhibiting CLN transcription.
Assuntos
Proteínas de Ciclo Celular , Ciclinas/fisiologia , Dano ao DNA , Proteínas Fúngicas/fisiologia , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/fisiologia , Alquilantes , Ciclo Celular/efeitos dos fármacos , Quinase do Ponto de Checagem 2 , DNA Fúngico/genética , Regulação para Baixo , Regulação Fúngica da Expressão Gênica , Metanossulfonato de Metila , Fosforilação , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The Swi6 transcription factor, required for G1/S-specific gene expression in Saccharomyces cerevisiae, is highly phosphorylated in vivo. Within the limits of resolution of the peptide analysis, the synchrony, and the time intervals tested, serine 160 appears to be the only site of phosphorylation in Swi6 that varies during the cell cycle. Serine 160 resides within a Cdc28 consensus phosphorylation site and its phosphorylation occurs at about the time of maximal transcription of Swi6- and Cdc28-dependent genes containing SCB or MCB elements. However, phosphorylation at this site is not Cdc28-dependent, nor does it control G1/S-specific transcription. The role of the cell cycle-regulated phosphorylation is to control the subcellular localization of Swi6. Phosphorylation of serine 160 persists from late G1 until late M phase, and Swi6 is predominantly cytoplasmic during this time. Aspartate substitution for serine 160 inhibits nuclear localization throughout the cycle. Swi6 enters the nucleus late in M phase and throughout G1, when serine 160 is hypophosphorylated. Alanine substitution at position 160 allows nuclear entry of Swi6 throughout the cell cycle. GFP fusions with the N-terminal one-third of Swi6 display the same cell cycle-regulated localization as Swi6.
