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Vol. 13, Issue 2, 480-492, February 2002
Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115
Fission yeast Rad3 is a member of a family of phosphoinositide
3-kinase -related kinases required for the maintenance of
genomic stability in all eukaryotic cells. In fission yeast, Rad3
regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures
Rad3 kinase activity in cells expressing physiological levels of the
protein. Using the assay, we demonstrate directly that Rad3 kinase
activity is stimulated by checkpoint signals. Of the five other G2/M
checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26
was required for Rad3 kinase activity. Because Rad26 has previously
been shown to interact constitutively with Rad3, our results
demonstrate that Rad26 is a regulatory subunit, and Rad3 is the
catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of
Rad26/Rad3 kinase activation in rad26.T12, a mutant that
is proficient for cell cycle arrest, but defective in recovery,
suggests that these two responses to checkpoint signals require
quantitatively different levels of kinase activity from the Rad3/Rad26 complex.
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