Hydroxyurea sensitivity reveals a role for ISC1 in the regulation of G₂/M

Saccharomyces cerevisiae cells lacking ISC1 (inositol phosphosphingolipase C) exhibit sensitivity to genotoxic agents such as methyl methanesulfonate and hydroxyurea (HU). Cell cycle analysis by flow cytometry revealed a G₂/M block in isc1cells when treated with methyl methanesulfonate or HU. Further investigation revealed that the levels of Cdc28 phosphorylated on Tyr-19, which plays an essential role in the regulation of the G₂/M checkpoint, were higher in synchronized and asynchronous cells lacking ISC1 in response to HU. Use of a Cdc28-Y19F mutant protected isc1from the G₂/M block. In wild type cells, HU induced a loss of the Swe1p kinase, the enzyme that phosphorylates Cdc28-Tyr-19, correlating with resumption of the cell cycle. In the isc1cells, however, the levels of Swe1p remained at sustained high levels in response to HU. Significantly, deletion of SWE1 in an isc1background overcame the G₂/M block in response to HU. The double isc1√swe1mutant also overcame the growth defect on HU. Taken together, these findings implicate Isc1p as an upstream regulator of Swe1p levels and stability and Cdc28-Tyr-19 phosphorylation, in effect signaling recovery from the effects of genotoxic stress and allowing G₂/M progression.