Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast.

Abstract

From yeast to humans, the cell cycle is tightly controlled by regulatory networks that regulate cell proliferation and can be monitored by dynamic visual markers in living cells. We have observed S phase progression by monitoring nuclear accumulation of the FHA-containing DNA binding protein Tos4, which is expressed in the G1/S phase transition. We use Tos4 localization to distinguish three classes of DNA replication mutants: those that arrest with an apparent 1C DNA content and accumulate Tos4 at the restrictive temperature; those that arrest with an apparent 2C DNA content, that do not accumulate Tos4; and those that proceed into mitosis despite a 1C DNA content, again without Tos4 accumulation. Our data indicate that Tos4 localization in these conditions is responsive to checkpoint kinases, with activation of the Cds1 checkpoint kinase promoting Tos4 retention in the nucleus, and activation of the Chk1 damage checkpoint promoting its turnover. Tos4 localization therefore allows us to monitor checkpoint-dependent activation that responds to replication failure in early vs. late S phase.