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

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 (;-H2AX), Chk2 (p T68 Chk2), and ATM (p S1981 ATM) 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. (

DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations

“…5 Chromosome instability in ICF syndrome has always been associated with hypomethylation of the involved chromosomal regions. 27 However, it is tempting to suggest that altered DNA replication may also contribute to the genomic instability that characterizes ICF cells, similar to what is observed in other pathological conditions (ie, cancer, 28 Bloom syndrome, 29 Rothmund-Thomson syndrome 30 ) where the alteration of the replication process is associated with a high level of spontaneously occurring and/or induced chromosome abnormalities.…”

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome

“…Asynchronous populations of cells were labeled by incubation for 40 min with 100 µM IdU (Sigma-Aldrich), washed with medium at 37 °C and then labeled by incubation for 40 min with 100 µM CldU (SigmaAldrich). The DNA solution was prepared as previously described 5 , and DNA was combed on silane-treated coverslips with a combing apparatus (Genomic Vision). Coverslips with combed DNA were baked overnight at 60 °C and incubated in 0.5 M NaOH-1 M NaCl solution for 15 min, with gentle shaking, for DNA denaturation.…”

“…In vitro, BLM unwinds DNA structures mimicking replication forks and homologous recombination intermediates 3 . The specific functions of BLM remain unclear, but it is widely thought that BLM is involved in restarting blocked replication forks 4,5 . In the absence of BLM, cells display a high rate of sister chromatid exchanges (SCEs), pathognomonic for BS 6 .…”

“…In the absence of BLM, cells display a high rate of sister chromatid exchanges (SCEs), pathognomonic for BS 6 . BS cells also display a slowing of replication fork progression associated with endogenous activation of the ATM-Chk2-γH2AX pathway 4,5 . This pathway is activated in precancerous lesions and is thought to be a part of an antitumorigenesis barrier 7,8 .…”

Defining replication origin efficiency using DNA fiber assays

“…These include overexpression of translesion DNA polymerases (Pillaire et al 2007), the depletion of CHK1 (Maya-Mendoza et al 2007), BLM (Rao et al 2007), HR proteins (Daboussi et al 2008), and topoisomerase I (Tuduri et al 2009) and exposure to HU (Ge et al 2007;Ibarra et al 2008). Importantly, the latter studies also revealed that dormant origins are dispensable for normal growth but are essential for viability under replication stress (Ge et al 2007;Ibarra et al 2008).…”