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DNA Damage and Cellular Stress Responses

Endogenous -H2AX-ATM-Chk2 Checkpoint Activation in Bloom's Syndrome Helicase–Deficient Cells Is Related to DNA Replication Arrested Forks

¹ Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland; ² Genome Stability Laboratory, Pasteur Institute, Paris, France; and ³ Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom

Requests for reprints: Yves Pommier, 37 Convent Drive, 37-5068, NIH, Bethesda, MD 20892. Phone: 301-496-5944; Fax: 301-402-0752. E-mail: pommier{at}nih.gov

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 (-H2AX), Chk2 (p^T68Chk2), and ATM (p^S1981ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of -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 -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. (Mol Cancer Res 2007;5(7):713–24)

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