A Novel Role for the Mitotic Spindle during DNA Segregation in Yeast: Promoting 2μm Plasmid-Cohesin Association

Abstract

The 2μm circle plasmid in Saccharomyces cerevisiae is a model for a stable, high-copy-number, extrachromosomal “selfish” DNA element. By combining a partitioning system and an amplification system, the plasmid ensures its stable propagation and copy number maintenance, even though it does not provide any selective advantage to its host. Recent evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation. We now demonstrate an unexpected and unconventional role for the mitotic spindle in the plasmid-partitioning pathway. The spindle specifies the nuclear address of the 2μm circle and promotes recruitment of the cohesin complex to the plasmid-partitioning locus STB. Only the nuclear microtubules, and not the cytoplasmic ones, are required for loading cohesin at STB. In cells recovering from nocodazole-induced spindle depolymerization and G₂/M arrest, cohesin-STB association can be established coincident with spindle restoration. This postreplication recruitment of cohesin is not functional in equipartitioning. However, normally acquired cohesin can be inactivated after replication without causing plasmid missegregation. In the mtw1-1 mutant yeast strain, the plasmid cosegregates with the spindle and the spindle-associated chromosomes; by contrast, a substantial number of the chromosomes are not associated with the spindle. These results are consistent with a model in which the spindle promotes plasmid segregation in a chromosome-linked fashion.

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A Novel Role for the Mitotic Spindle during DNA Segregation in Yeast: Promoting 2μm Plasmid-Cohesin Association

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

This work was supported by funds from the National Institutes of Health (GM64363). S.M. received partial support through the William Livingston Fellowship from the University of Texas at Austin.

We are grateful to David Botstein, Douglas Koshland, Sue Biggins, and Vincent Guacci for providing strains and reagents.