Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling

DNA Repair (Amst). 2008 Mar 1;7(3):405-17. doi: 10.1016/j.dnarep.2007.11.011. Epub 2008 Jan 8.

Abstract

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia / metabolism
  • Ataxia Telangiectasia / pathology
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Damage / radiation effects*
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Fibroblasts / radiation effects
  • Fluorescent Antibody Technique
  • G1 Phase / physiology*
  • G1 Phase / radiation effects
  • Genes, cdc / physiology*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Infrared Rays
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Morpholines / pharmacology
  • Nijmegen Breakage Syndrome / metabolism
  • Nijmegen Breakage Syndrome / pathology
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphorylation / radiation effects
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Pyrones / pharmacology
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism
  • Tumor Suppressor Proteins / antagonists & inhibitors
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism
  • Tumor Suppressor p53-Binding Protein 1
  • X-Rays

Substances

  • 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • Morpholines
  • NBN protein, human
  • Nuclear Proteins
  • Pyrones
  • TP53BP1 protein, human
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Tumor Suppressor p53-Binding Protein 1
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • DNA Repair Enzymes