title: 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.
authors.affiliation: Molecular and Computational Biology, University of Southern California, Los Angeles, California, USA. sabatino@usc.edu
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Sabatinos
authors.forename: Marc D
authors.initials: MD
authors.lastname: Green
authors.forename: Susan L
authors.initials: SL
authors.lastname: Forsburg
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D064148:Minichromosome Maintenance Complex Component 4; D008954:Models, Biological; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C533276:MRC1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D064148:Minichromosome Maintenance Complex Component 4; C078490:mcm4 protein, S pombe; D006918:Hydroxyurea