RESUMEN
CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.
Asunto(s)
Cromátides/fisiología , Cromosomas Fúngicos/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Homeodominio , Proteínas , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animales , Replicación del ADN , ADN de Hongos , ADN Ribosómico , Proteínas de Unión al ADN/genética , Células Eucariotas/metabolismo , Proteínas Fúngicas/genética , Fase G2 , Humanos , Antígenos de Histocompatibilidad Menor , Mitosis/fisiología , Mutagénesis , Proteína de Replicación C , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/metabolismo , Huso Acromático/fisiologíaRESUMEN
We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations. To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26. ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1. These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants. In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.