Characterization of Schizosaccharomyces pombe Hus1: a PCNA-Related Protein That Associates with Rad1 and Rad9

doi:10.1128/MCB.20.4.1254-1262.2000

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Molecular and Cellular Biology, February 2000, p. 1254-1262, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of Schizosaccharomyces pombe Hus1: a PCNA-Related Protein That Associates with Rad1 and Rad9

Thomas Caspari,¹ Maria Dahlen,² Gunilla Kanter-Smoler,² Howard D. Lindsay,¹ Kay Hofmann,³ Konstantinos Papadimitriou,¹ Per Sunnerhagen,² and Antony M. Carr¹^,^*

MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, United Kingdom¹; Department of Molecular Biology, Lundberg Laboratory, Göteborg University, S-413 90 Göteborg, Sweden²; and MEMOREC Stoffel GmbH, D-50829 Cologne, Germany³

Received 11 August 1999/Returned for modification 21 September 1999/Accepted 12 November 1999

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-taggedHus1 reveals four discrete forms. The main form, Hus1-B, participatesin a protein complex with Rad9 and Rad1, consistent with reportsthat Rad1-Hus1 immunoprecipitation is dependent on the rad9⁺ locus. A small proportion of Hus1-B is intrinsically phosphorylatedin undamaged cells and more becomes phosphorylated after irradiation.Hus1-B phosphorylation is not increased in cells blocked in earlyS phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-Bcomplex is readily detectable, but upon cofractionation of solubleextracts, the majority of each protein is not present in thiscomplex. Indirect immunofluorescence demonstrates that Hus1 isnuclear and that this localization depends on Rad17. We show thatRad17 defines a distinct protein complex in soluble extracts thatis separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction,in vitro association and in vivo overexpression experiments suggesta transient interaction between Rad1 andRad17.

^* Corresponding author. Mailing address: MRC Cell Mutation Unit, Brighton BN1 9RR, United Kingdom. Phone: 0044-1273-678-122.Fax: 0044-1273-678-121. E-mail: a.m.carr{at}sussex.ac.uk.

Molecular and Cellular Biology, February 2000, p. 1254-1262, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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