Rolle des Sgst1/Top3/Rmi1-Komplexes bei der Reparatur steckengebliebener Replikationsgabeln in der Hefe Saccharomyces Cerevisiae
Zusammenfassung der Projektergebnisse
The Saccharomyces cerevisiae gene SGS1 has been studied extensively worldwide m recent years due to its potential to inform us about the role of its human orthologues, which are involved in suppression of cancer and premature aging. Sgs1p belongs to the family of RecQ helicases that are conserved in evolution and act to maintain genome stability by regulating the process of homologous recombination. Sgs1p is involved in the repair of replication forks that stalled either for topological reasons (DNA adducts or DNA secondary structures) or due to single-stranded breaks or gaps. Sgs1p is part of a heterotrimeric protein complex, along with Top3p, the sole S. cerevisiae type IA topoisomerase, and Rmi1p (also known as Nce4p), a DNA-binding protein. Deletion of the TOP3 or the RMI1 gene causes a slow growth phenotype that is suppressed by deletion of SGS1. It has been hypothesized that Sgs1p creates a DNA processing intermediate that is toxic in the absence of Top3p or Rmi1p function. S. cerevisiae strains that lack either Top3p or Rmi1p have a low viability and a reduced spore viability. As a consequence those strains readily accumulate second-site suppressor mutations, especially mutations in SGS1. This makes and rmi1∆ strains difficult to work with as the accumulation of suppressor mutations complicates the interpretation of experimental data. The host laboratory had shown that overexpression of the dominant negative Top3p mutant Top3Y356F generates a top3∆-like phenotype. By overexpressing Top3Y356F from a plasmid containing an inducible promoter it is therefore possible to inducibly delete Top3p function. This system facilitates the analysis of the consequences of Top3p loss while reducing the number of suppressor mutations. One of the aims of my research project was to establish a system to inducibly delete Rmi1p function. I screened for dominant negative Rmi1p mutants but I did not identify a mutant that could generate an rmiI∆-like phenotype. I then performed a screen for Rmi1p mutants, that are functional at 25°C but non-functional at 35°C, so called temperature-sensitive mutants. I successfully identified two such mutant proteins and integrated the respective rmil alleles into the genome of an S. cerevisiae strain. The resulting temperature-sensitive yeast strains have a wild type-like phenotype at 25°C but upon shifting the temperature to 35°C show an rmiI∆-like phenotype. As a second project I screened for high-copy suppressors of 7op3∆-induced slow growth using the system to inducibly delete Top3p function outlined above. High-copy suppressors frequently encode proteins that form a complex with the target protein or which act in the same pathway. I was able to identify to possible high-copy suppressors of Top3Y356F-induced slow growth. MIG1 and PIF1. The MIG1 gene is not a true high-copy suppressor of Top3Y336F-induced slow growth slow growth at it suppresses gene expression from the inducible promoter that was used to control Top3Y356F expression. The identification of MIG1 as a hit in the high-copy suppressor screen validates the approach to use Top3Y356F overexpression in order to mimic a top3∆-like phenotype. The PIF1 gene, which encodes a DNA helicase, turned out to be a true suppressor of Top3Y356F-induced slow growth.
