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Molecular and Cellular Biology, January 2005, p. 767-778, Vol. 25, No. 2
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.2.767-778.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of the Kinetochore DASH Complex Reveals an Antagonistic Relationship with the Ras/Protein Kinase A Pathway and a Novel Subunit Required for Ask1 Association

Ju-mei Li,1,2,{dagger} Yumei Li,3,{dagger} and Stephen J. Elledge1,2,3*

Department of Genetics, Harvard Medical School, Center for Genetics and Genomics, Brigham and Women's Hospital, Boston, Massachusetts,1 Department of Molecular and Human Genetics,2 Verna and Marrs McLean Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas3

Received 20 September 2004/ Returned for modification 14 October 2004/ Accepted 18 October 2004

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.

* Corresponding author. Mailing address: Harvard Medical School, 77 Ave. Louis Pasteur, Boston, MA 02115. Phone: (617) 525-4510. Fax: (617) 525-4500. E-mail: selledge{at}genetics.med.harvard.edu.

{dagger} J.L. and Y.L. contributed equally to this study.

Molecular and Cellular Biology, January 2005, p. 767-778, Vol. 25, No. 2
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.2.767-778.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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