The decision to initiate division is very important, as once cells have initiated division they are committed to complete it. In Saccharomyces cerevisiae, commitment to a new round of cell division occurs at a regulatory point in late G1 called START. Progression through START requires the activation of the cyclin dependent kinase Cdc28p by the G1 cyclins. G1 cyclins in complex with Cdc28p activate the transcription of approximately 100 genes involved in the G1 to S transition and degradation of Sic1p, an inhibitor of B type cyclins, and thus are important for initiation of DNA replication. Despite the widely studied role of regulatory cyclins and cyclin dependent kinase in the G1 to S transition, how cells determine when to initiate DNA replication is poorly understood. We have identified several gene products, which when overexpressed, cause cells to initiate DNA replication faster than wild type. Here we discuss the role of DCR2 (Dosage dependent Cell cycle Regulator), GID8 (Glucose Induced Degradation) and KEM1 (Kar-Enhancing Mutation) in the regulation of START. Over expression of DCR2 and GID8 accelerates initiation of DNA replication. Cells lacking both these genes delay initiation of DNA replication. Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote START. Further, we show that DCR2, which codes for a metallo-phosphoesterase, might regulate completion of START by affecting degradation of Sic1p. Over expression of DCR2 lowers the half-life of Sic1p without altering the expression of Cln2p. The evidence suggests that Dcr2p affects START completion through dephosphorylation of Sic1p. KEM1 is a Saccharomyces cerevisiae gene, conserved in all eukaryotes, which codes for a 5’-3’ cytoplasmic exonuclease. This exonuclease is involved in exiting mitosis, by degrading the mRNA of the mitotic cyclin CLB2. Besides its role in mitotic exit, an enzymatically inactive version of Kem1p can accelerate the G1 to S transition and initiation of DNA replication when over expressed. This result suggests that Kem1p might have a previously unrecognized role in the G1 to S transition independent of its exonuclease activity, and supports the notion that Kem1p is a multifunctional protein with distinct and separable roles.

Orientador: Cleslei Fernando Zanelli / Resumo: eIF5A é um fator de elongação da tradução conservado em arqueias e eucariotos, que interage com a subunidade maior do ribossomo 80S. É a única proteína que possui o aminoácido hipusina, formado pós-traducionalmente, essencial para sua função. eIF5A auxilia na tradução de proteínas que contêm motifs ricos em prolina, pois sequência repetida deste aminoácido pode causar parada do ribossomo em tradução devido à rigidez estrutural que forma. Na ausência de eIF5A funcional ocorre parada do ciclo celular em G1, na transição de G1 para S, tanto em Saccharomyces cerevisiae quanto em mamíferos, sugerindo papel essencial desta na tradução de proteínas importantes para a progressão do ciclo celular. Neste trabalho foi proposta a identificação das proteínas relevantes para a fase G1 que se encontram diferencialmente presentes durante desativação de eIF5A em S. cerevisiae. Foram utilizados dados de larga escala de perfil proteômico diferencial entre linhagens selvagens e mutantes de eIF5A, previamente obtido por nosso grupo, como ponto de partida para identificação das proteínas relevantes. A confirmação de níveis diferenciais destas proteínas foi realizada utilizando metodologia de Western blot após indução da desativação de eIF5A, utilizando o mutante sensível a temperatura hyp2-3. A desativação foi feita através do cultivo da linhagem mutante em temperatura semi-permissiva, que induz perda da função de eIF5A, porém mantendo viabilidade celular. Foi observado que a proteína Cka2 está di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: eIF5A is a translation elongation factor conserved in archaea and eukaryotes, that interacts with the major subunit of the 80S ribosome. It is the only protein to contain the amino acid hypusine, formed post-translationally, essential to its function. eIF5A helps the translation of proteins that contain proline enriched motifs, as repeated sequence of this amino acid can cause ribosome stall during translation due to the rigid structure that it forms. In the absence of functional eIF5A occurs cell cycle arrest in G1, on the transition of G1 to S, both in Saccharomyces cerevisiae and mammals, suggesting an essential role of it on the translation of important proteins to the progression of the cell cycle. In this work, it was proposed the identification of relevant proteins to the G1 phase that are present differentially during deactivation of eIF5A in S. cerevisiae. It was used data of high-throughput differential proteomic profile between wild type strains and eIF5A mutant strains, previously obtained by our group, as a starting point to the identification of the relevant proteins. The confirmation of the differential levels of these proteins was made using Western blot methodology after induction of eIF5A deactivation, using the temperature-sensitive mutant hyp2-3. The deactivation was performed through cultivation of the mutant strain in semipermissive temperature, that induces loss of function of eIF5A but maintain cell viability. It was observed that the protein Cka2 is d... (Complete abstract click electronic access below) / Mestre