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Schizosaccharomyces pombe Orc5 plays multiple roles in the maintenance of genome stability throughout the cell cycle

Hiroaki Kato, Fujihiko Matsunaga, Shota Miyazaki, Ling Yin, Gennaro D’Urso, Katsunori Tanaka and Yota Murakami

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The six-subunit origin recognition complex (ORC) acts as a landing pad for factors that initiate DNA replication by binding to replication origins. In addition, ORC is involved in other processes such as transcriptional gene silencing and sister chromatid cohesion in Saccharomyces cerevisiae. However, whether these functions of ORC are specific to Saccharomyces cerevisiae or are shared by the ORC of other organisms is currently unclear. Analysis of two temperature-sensitive alleles of the fifth ORC subunit of Saccharomyces pombe, orc5-H19 and orc5-H37, indicates that Orc5 of Saccharomyces pombe has similar multiple functions to those of Orc5 of Saccharomyces cerevisiae. The orc5-H19 cells were defective in DNA replication initiation, and execution point analysis of this mutant revealed that ORC functions before metaphase to prepare for the initiation of replication in the next cell cycle. The orc5-H37 cells seemed to complete DNA synthesis but were arrested before entering M phase. In both mutants, the rads-chk1 checkpoint was activated to prevent mitosis, suggesting that this checkpoint pathway monitors the functional integrity of ORC. In addition, orc5-H37 cells showed premature separation of sister chromatids, which resulted in cell growth being dependent on the mad2-dependent spindle checkpoint. Consistently, this mutant showed a defect in the loading of Rad21, a cohesin component. Based on these observations, we propose that Orc5 has at least two distinct functions that can be separated genetically. Taken together, our results provide further support for the idea that ORC plays multiple functions during the cell cycle.

Authors

Hiroaki Kato

Kyoto University; Kyoto, Japan

Fujihiko Matsunaga

Kyoto University; Kyoto, Japan

Shota Miyazaki

Kyoto University; Kyoto, Japan

Ling Yin

University of Miami School of Medicine; Miami, FL

Gennaro D’Urso

University of Miami School of Medicine; Miami, FL

Katsunori Tanaka

Shimane University; Matsue, Shimane, Japan

Yota Murakami

Kyoto University; Kyoto, Japan

Purchase article for $19

Subscribe to this journal for $129/year