Characterization of Schizosaccharomyces pombeHus1: a PCNA-Related Protein That Associates with Rad1 and Rad9

Abstract

Hus1 is one of six checkpoint Rad proteins required for allSchizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9⁺ locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1–Rad9–Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.

ACKNOWLEDGMENTS

We thank Per Uhrskov Christensen for reading the manuscript and helpful discussions.

T.C. was sponsored by fellowship Ca197/2-1 from the Deutsche Forschungsgemeinschaft (Bonn, Germany). A.M.C. and H.D.L. are supported by the MRC and Euratom contract F14PCT950010. P.S. acknowledges the Swedish Cancer Fund (grant 2163-B97-08XAC) and Swedish Radiation Protection Institute (grant 1092.98).