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The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer predisposing Bloom's syndrome (BS). Studies with BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (γ-H2AX), Chk2 (P-T68-Chk2) and ATM (P-S1981-ATM) in interphase cells. Interestingly, the mitotic fraction of γ-H2AX foci did not appear to be higher in BLM deficient cells. Pulse-labeling with Iodo-deoxyuridine and immunofluorescence microscopy showed colocalization of γ-H2AX, ATM and Chk2 together with replication foci. These foci co-stained 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 elevated frequency of origin firing and reduced average fork velocity compared to BLM-proficient cells. These studies reveal direct replication defects and their association with endogenously activated ATM and Chk2 in BLM-deficient cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA