Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

DNA Repair (Amst). 2011 Aug 15;10(8):815-25. doi: 10.1016/j.dnarep.2011.04.031. Epub 2011 May 23.


To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ɛ and Ddc1 checkpoint protein. Labeling of DNA polymerase ɛ catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cell Cycle Proteins / chemistry*
  • DNA Breaks, Single-Stranded*
  • DNA Polymerase II / chemistry*
  • DNA Repair*
  • DNA, Fungal / chemistry*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Protein Binding
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Staining and Labeling


  • Cell Cycle Proteins
  • DNA, Fungal
  • Ddc1 protein, S cerevisiae
  • Peptide Fragments
  • Saccharomyces cerevisiae Proteins
  • DNA Polymerase II