Continued DNA synthesis in replication checkpoint mutants leads to fork collapse. Molecular and cellular biology 32

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 andMrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1 andmrc1 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 re-lease. 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, cds1 andmrc1 forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release fromHU block. Fission yeast Schizosaccharomyces pombe cells treated with hy-droxyurea (HU) suffer nucleotide depletion and activate the intra-S phase checkpoint. This causes reversible cell cycle arrest in early S phase and prevents firing of late replication origins (12, 19, 62). The intra-S phase checkpoint stabilizes existing DNA replica-tion forks and promotes recovery and replication resumption upon release. Loss of the intra-S phase checkpoint corrupts repli-cation forks, leading toDNAdamage and cell death. However, th