Continued DNA synthesis in replication checkpoint mutants leads to fork collapse

Mol Cell Biol. 2012 Dec;32(24):4986-97. doi: 10.1128/MCB.01060-12. Epub 2012 Oct 8.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Checkpoint Kinase 2
  • DNA Damage
  • DNA Replication / drug effects
  • DNA Replication / genetics
  • DNA, Fungal / biosynthesis*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Genes, Fungal
  • Hydroxyurea / pharmacology
  • Minichromosome Maintenance Complex Component 4
  • Models, Biological
  • Mutation
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Schizosaccharomyces / drug effects
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism

Substances

  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • MRC1 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • Checkpoint Kinase 2
  • Cds1 protein, S pombe
  • Protein Serine-Threonine Kinases
  • Minichromosome Maintenance Complex Component 4
  • mcm4 protein, S pombe
  • Hydroxyurea