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

Abstract

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.

ACKNOWLEDGMENTS

We thank D. Gilbert, T. Yonesaki, H. Araki, J. T. Lee, C. Obuse, and T. Tsurimoto for critical reading of the manuscript and helpful discussions; M. Yanagida, P. Nurse, and B. Stillman for providing yeast strains and plasmids; T. Tanaka for advice on chromatin immunoprecipitation analysis; and T. Usui for advice on preparation of spread nucleoids.

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.