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Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint
Author(s) -
Lehmann Anna,
Katayama Satoshi,
Harrison Clare,
Dhut Susheela,
Kitamura Kenji,
McDonald Neil,
Toda Takashi
Publication year - 2004
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1356-9597.2004.00730.x
Subject(s) - skp1 , cullin , biology , g2 m dna damage checkpoint , f box protein , ubiquitin ligase , microbiology and biotechnology , cell cycle checkpoint , ubiquitin , cell cycle , genetics , cell , gene
Skp1 is a central component of the E3 ubiquitin ligase SCF ( S kp1‐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 pof3Δ and skp1 ts , implicating a novel role for Skp1 in the checkpoint‐signalling cascade.