Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks

Mol Cancer Res. 2007 Jul;5(7):713-24. doi: 10.1158/1541-7786.MCR-07-0028.

Abstract

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Adenosine Triphosphatases / deficiency*
  • Ataxia Telangiectasia Mutated Proteins
  • Bloom Syndrome / enzymology
  • Bloom Syndrome / genetics
  • Bloom Syndrome / pathology*
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Transformed
  • Checkpoint Kinase 2
  • DNA Helicases / deficiency*
  • DNA Replication*
  • DNA Topoisomerases, Type I / metabolism
  • DNA-Binding Proteins / metabolism*
  • Histones / metabolism*
  • Humans
  • Metaphase
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Transport
  • Rad51 Recombinase / metabolism
  • RecQ Helicases
  • Recombination, Genetic / genetics
  • Replication Origin
  • Tumor Suppressor Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • Phosphoproteins
  • Tumor Suppressor Proteins
  • Checkpoint Kinase 2
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK2 protein, human
  • Protein Serine-Threonine Kinases
  • RAD51 protein, human
  • Rad51 Recombinase
  • Adenosine Triphosphatases
  • Bloom syndrome protein
  • DNA Helicases
  • RecQ Helicases
  • DNA Topoisomerases, Type I