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¹ Laboratory of Cell Regulation,
² Laboratory of Structural Biology, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

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. skp1^ts 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 pof3 and skp1^ts, implicating a novel role for Skp1 in the checkpoint-signalling cascade.

Present address: ^aAnalytical Research Centre for Experimental Sciences, Saga University, Saga 840-8502 Japan,

^bCentre for Gene Science, Hiroshima University, 1-4-2 Kagamiyama, Higashi-Hiroshima 739-8537, Japan

^* Correspondence: E-mail: toda{at}cancer.org.uk

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