Open AccessCharacterization of Schizosaccharomyces pombeHus1: a PCNA-Related Protein That Associates with Rad1 and Rad9
Author(s) -
Thomas Caspari,
Maria Dahlén,
Gunilla Kanter-Smoler,
Howard D. Lindsay,
Kay Hofmann,
Konstantinos Papadimitriou,
Per Sunnerhagen,
Antony Carr
Publication year - 2000
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.20.4.1254-1262.2000
Subject(s) - biology , immunoprecipitation , schizosaccharomyces pombe , schizosaccharomyces , proliferating cell nuclear antigen , saccharomyces cerevisiae , microbiology and biotechnology , protein–protein interaction , dna binding protein , dna , biochemistry , yeast , transcription factor , gene
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 therad9 + 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.