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Updated: Aug 17, 2026
Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins
Published on: September 28, 2012
Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint
Anna Lehmann1, Satoshi Katayama, Clare Harrison
1Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Labratories, 44 Lincoln's Inn Fields, London WC2A 3PA, UK.
Abstract:
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.
Insights
Skp1, a key component of the SCF ubiquitin ligase, was studied using temperature-sensitive mutants. These mutants revealed Skp1
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- Skp1 is a crucial adapter protein in the SCF (Skp1-Cullin-1-F-box) E3 ubiquitin ligase complex.
- It bridges Cullin-1 and F-box proteins, which determine substrate specificity.
Purpose of the Study:
- To investigate the role of Skp1 in SCF complex function and cellular processes.
- To characterize the impact of Skp1 mutations on F-box protein binding and cell cycle regulation.
Main Methods:
- Isolation of temperature-sensitive (ts) Skp1 mutants using mutagenic PCR.
- Analysis of Skp1 mutant binding affinities to F-box proteins and Cullin-1 (Pcu1) via co-immunoprecipitation.
- Assessment of cell cycle progression and DNA damage checkpoint activation in skp1(ts) cells.
Main Results:
- Nine independent ts Skp1 mutants were identified.
- Mutant Skp1 retained binding to Pcu1 but showed reduced binding to F-box proteins Pof1, Pof3, and Pof10.
- skp1(ts) cells displayed a G2 cell cycle delay due to DNA damage checkpoint activation.
- Checkpoint abrogation in skp1(ts) cells suppressed the G2 delay and rescued the ts phenotype, unlike in pof3 mutants.
Conclusions:
- Skp1 plays a critical role in regulating F-box protein interactions within the SCF complex.
- Skp1 is implicated in the DNA damage checkpoint signaling pathway, suggesting a novel function beyond its adapter role.
- The mechanism of DNA damage checkpoint activation differs between Skp1 mutants and pof3 mutants.
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