Two checkpoint complexes are independently recruited to sites of DNA damage in vivo.
The Ddc1/Rad17/Mec3 complex and Rad24 are DNA damage checkpoint components with limited homology to replication factors PCNA and RF-C, respectively, suggesting that these factors promote checkpoint activation by "sensing" DNA damage directly. Mec1 kinase, however, phosphorylates the checkpoint protein Ddc2 in response to damage in the absence of all other known checkpoint proteins, suggesting instead that Mec1 and/or Ddc2 may act as the initial sensors of DNA damage. In this paper, we show that Ddc1 or Ddc2 fused to GFP localizes to a single subnuclear focus following an endonucleolytic break. Other forms of damage result in a greater number of Ddc1-GFP or Ddc2-GFP foci, in correlation with the number of damage sites generated, indicating that Ddc1 and Ddc2 are both recruited to sites of DNA damage. Interestingly, Ddc2 localization is severely abrogated in mec1 cells but requires no other known checkpoint genes, whereas Ddc1 localization requires Rad17, Mec3, and Rad24, but not Mec1. Therefore, Ddc1 and Ddc2 recognize DNA damage by independent mechanisms. These data support a model in which assembly of multiple checkpoint complexes at DNA damage sites stimulates checkpoint activation. Further, we show that although Ddc1 remains strongly localized following checkpoint adaptation, many nuclei contain only dim foci of Ddc2-GFP, suggesting that Ddc2 localization may be down-regulated during resumption of cell division. Lastly, visualization of checkpoint proteins localized to damage sites serves as a useful tool for analysis of DNA damage in living cells.
Melo
J A
JA
Mt. Zion Cancer Research Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94115, USA.
Cohen
J
J
Toczyski
D P
DP
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008954:Models, Biological; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C440013:Ddc1 protein, S cerevisiae; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C501395:RAD17 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases