Abstract
Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS.
Highlights
Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers
We previously demonstrated that I-SceIinduced double-strand breaks (DSBs) in human cells were predominantly repaired by nonhomologous end joining (NHEJ), which resulted in small deletions; repairs via homologous recombination (HR) were minimal, even during the late S/G2 phases [16, 17]
Based on the role of BLM helicase in HR and DSB repair, we propose that the mutator phenotype and genomic instability caused by BLM helicase deficiency result in large-scale loss of heterozygosity (LOH)
Summary
Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS. A positive-negative selection of TK phenotypes and LOH analysis using the TK6 cell assay system can elucidate NHEJ and HR repair mechanisms Based on this assay system, we successfully introduced a meganuclease I-SceI site into the TK gene [15]. In addition to these clinical manifestations, BS cells exhibit a number of cytogenetic abnormalities, including
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