Lehmann, Anna; (2004) Molecular analysis of fission yeast Skp1, a core component of the Scf ubiquitin ligase. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (16MB)

Abstract

Skp1 is a central component of the E3 ubiquitin ligase, Skp1, Cullin, F-box or SCF. It forms an adaptor bridge between a rigid cullin and the substrate specificity-determining component, the F-box. S. pombe skp1+ was identified from the sequenced fission yeast genome. A deletion of the full ORF proved to be lethal as were deletions of other core components of this ligase. In order to consider a cellular role for Skp1 a temperature sensitive mutant screening analysis was carried out. All the mutants created from this screen were sequenced and the resulting sequences showed that the alleles fell into five classes of skp1ts mutants. Three different point mutations were obtained as were two mutations causing read through of the stop codon. Modelling of these mutations on the published structure of human SKP1 suggested that these mutations would affect F-box binding but not binding to the core SCF through Pcu1 the fission yeast cullin known to be involved in this complex. Initial analysis demonstrated that all five types of skp1ts mutant displayed similar phenotypes. Therefore one mutant allele, showing the most severe phenotype (skp1tsA7) was chosen for further detailed characterisation. This characterisation revealed that skp1tsA7 cells are elongated at the non-permissive temperature. This elongation can be attributed to a G2 cell cycle delay caused by activation of the DNA damage checkpoint. skp1ts A7 cells also show abnormal chromosome segregation possibly arising from an inability to properly elongate their spindle at the restrictive temperature. Given the results from speculative modelling, further analysis of the binding capabilities of this skp1ts A7 mutant were analysed. This showed that Skp1ts A7 was able to retain binding to Pcu1 and the majority of F-box proteins, however, binding to two F-box proteins, Pof3, an F-box involved in maintaining genome integrity and Pof1, an essential F-box, were abrogated. The data presented in this thesis show the characterisation of skp1ts A7 phenotype and attempt to understand the aspects of the phenotype in the light of the interactions affected and the role for proteolysis in the cell cycle.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item