RESUMO
The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Delta cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Delta rrm3Delta cells also have a severe growth defect and accumulate DNA damage in late S/G2. In contrast, esc2Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Delta cells.
Assuntos
Cromátides/genética , Genoma Fúngico , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Ciclo Celular , Proteínas de Ciclo Celular , Segregação de Cromossomos , Dano ao DNA , Replicação do DNA , Deleção de Genes , Inativação Gênica , Hidroxiureia/farmacologia , Modelos Genéticos , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.
Assuntos
Adenosina Trifosfatases/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Sequência de Bases , DNA Helicases/genética , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Instabilidade Genômica , Lisina/química , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Early in eukaryotic cell cycle, a pre-RC is assembled at each replication origin with ORC, Cdc6, Cdt1 and Mcm2-7 proteins to license the origin for use in the subsequent S phase. Licensed origin must then be activated by S-Cdk and Ddk. At the onset of S phase, RPA is loaded on to the ARS in a reaction stimulated by S-Cdk and Ddk, followed by Cdc45-dependent loading of pol alpha, -delta, and -epsilon. This study examines cell cycle-dependent localization of pol alpha, -delta and -epsilon in Saccharomyces cerevisiae using immuno-histochemical and chromatin immuno-precipitation methods. The results show that pol alpha, -delta, or -epsilon localizes on chromatin as punctate foci at all stages of the cell cycle. However, some foci overlap with or are adjacent to foci pulse-labeled with bromodeoxyuridine during S phase, indicating these are replicating foci. DNA microarray analysis localized pol alpha, -delta, and -epsilon to early firing ARSs on yeast chromosome III and VI at the beginning of S phase. These data collectively suggest that bidirectional replication occurs at specific foci in yeast chromosomes and that pol alpha, -delta, and -epsilon localize and function together at multiple replication forks during S phase.
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Replicação do DNA/fisiologia , Saccharomyces cerevisiae/enzimologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Imunoprecipitação da Cromatina , Cromossomos Fúngicos/enzimologia , Cromossomos Fúngicos/genética , DNA Polimerase I/genética , DNA Polimerase II/genética , DNA Polimerase beta/genética , Replicação do DNA/genética , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Saccharomyces cerevisiae/genéticaRESUMO
Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.