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Cdc45 protein-single-stranded DNA interaction is important for stalling the helicase during replication stress.

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.

Pubmed ID: 23382391

Authors

  • Bruck I
  • Kaplan DL

Journal

The Journal of biological chemistry

Publication Data

March 15, 2013

Associated Grants

None

Mesh Terms

  • Alleles
  • Anisotropy
  • Biotin
  • Cell Separation
  • DNA Helicases
  • DNA Replication
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • DNA-Directed DNA Polymerase
  • Flow Cytometry
  • Gene Expression Regulation, Fungal
  • Glutathione Transferase
  • Hydroxyurea
  • Microscopy, Fluorescence
  • Models, Biological
  • Mutation
  • Nuclear Proteins
  • Phosphorylation
  • Saccharomyces cerevisiae Proteins
  • Saccharomycetales