Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint.
Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.
Lehmann
Anna
A
Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Labratories, 44 Lincoln's Inn Fields, London WC2A 3PA, UK.
Katayama
Satoshi
S
Harrison
Clare
C
Dhut
Susheela
S
Kitamura
Kenji
K
McDonald
Neil
N
Toda
Takashi
T
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism; D004249:DNA Damage; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase; D018816:Genes, cdc; D016681:Genome, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D044783:F-Box Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D044843:SKP Cullin F-Box Protein Ligases; C120507:SKP1 protein, S cerevisiae; C504781:SKP1 protein, S pombe