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Molecular and Cellular Biology, October 1999, p. 7228-7236, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

Yuya Ogawa,1,dagger Tatsuro Takahashi,1 and Hisao Masukata1,2,*

Department of Biology, Graduate School of Science, Osaka University,1 and PRESTO, Japan Science and Technology Corporation,2 Toyonaka, Osaka 560-0043 Japan.

Received 20 May 1999/Returned for modification 18 June 1999/Accepted 9 July 1999

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1+/cdc19+ and mis5+ genes, respectively, were associated with chromatin DNA only during the G1 and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G1 and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G1 and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

* Corresponding author. Mailing address: Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Phone: 81-6-6850-5432. Fax: 81-6-6850-5440. E-mail: masukata{at}bio.sci.osaka-u.ac.jp.

dagger Present address: Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02114.

Molecular and Cellular Biology, October 1999, p. 7228-7236, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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