Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related proteinthat associates with Rad1 and Rad9

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharom yces 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 dep endent on the rad9(+) locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irr adiation. 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 extrac ts, the majority of each protein is not present in this complex. Indirect i mmunofluorescence demonstrates that Hus1 is nuclear and that this localizat ion depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, t wo-hybrid interaction, in vitro association and in vivo overexpression expe riments suggest a transient interaction between Rad1 and Rad17.