Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Abstract

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01060-12.

ACKNOWLEDGMENTS

We thank JiPing Yuan for technical assistance, Forsburg laboratory members for beneficial discussion, and anonymous reviewers who made helpful suggestions during the review process. We thank Beth Sullivan for assistance with DNA fiber methods, Toshio Tsukiyama for helpful discussion, and Johanne Murray, Oscar Aparcio, and Matthew Michael for critical reading of the manuscript.

This work was supported by grant NIH R01 GM059321 to S.L.F.