Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response

Nucleic Acids Res. 2000 Oct 1;28(19):3725-32. doi: 10.1093/nar/28.19.3725.

Abstract

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle* / drug effects
  • Cell Cycle* / radiation effects
  • Chromosomes, Fungal / drug effects
  • Chromosomes, Fungal / genetics
  • Chromosomes, Fungal / metabolism
  • Chromosomes, Fungal / radiation effects
  • Cyclin B / genetics
  • Cyclin B / metabolism
  • DNA Damage / drug effects
  • DNA Damage / genetics
  • DNA Damage / radiation effects
  • DNA Repair
  • DNA Replication / drug effects
  • DNA Replication / radiation effects
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism*
  • Dose-Response Relationship, Radiation
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Genes, Fungal / genetics
  • Glycosyltransferases / metabolism
  • Humans
  • Hydroxyurea / pharmacology
  • Intracellular Signaling Peptides and Proteins
  • Mutation / genetics
  • Nuclear Proteins
  • Phosphorylation / drug effects
  • Phosphorylation / radiation effects
  • Protein-Serine-Threonine Kinases / metabolism
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins*
  • Saccharomyces cerevisiae* / cytology
  • Saccharomyces cerevisiae* / drug effects
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / radiation effects
  • Telomere / drug effects
  • Telomere / genetics
  • Telomere / metabolism
  • Telomere / radiation effects
  • Transcription Factors*
  • Ultraviolet Rays

Substances

  • Cyclin B
  • DNA, Fungal
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Fungal Proteins
  • Intracellular Signaling Peptides and Proteins
  • Nuclear Proteins
  • RFA2 protein, S cerevisiae
  • RPA1 protein, human
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Glycosyltransferases
  • DNA-Activated Protein Kinase
  • MEC1 protein, S cerevisiae
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • TEL1 protein, S cerevisiae
  • Hydroxyurea