Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells

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.

BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes

“…DNA repair via HR can also result in LOH when the homologous chromosome is used for repair instead of the sister chromatid (Moynahan and Jasin, 1997). It has previously been shown that BLM deficient cells display high levels of LOH (LaRocque et al, 2011;Luo et al, 2000;Suzuki et al, 2016). However, these results were obtained using systems that rely on selection of cells that underwent LOH at a specific locus.…”

“…However, if an exchange event occurs between homologs instead of sister chromatids, this can lead to loss of heterozygosity (LOH) (Moynahan and Jasin, 1997). LOH is a known driver of cancer, and it has previously been shown that BLM deficient cells display elevated LOH levels (LaRocque et al, 2011;Luo et al, 2000;Suzuki et al, 2016).…”

“…BS cells display higher numbers of gH2Ax foci (Rao et al, 2005), indicating frequent activation of the DNA damage response in the absence of BLM. It has also been shown that BS cells display elevated levels of loss of heterozygosity (LOH), due to exchanges between homologous chromosomes (LaRocque et al, 2011;Luo et al, 2000;Suzuki et al, 2016).…”

Use of E18 Cell Model to Quantify DNA Strand Break Associated Bystander Effect (DSB-ABE)

“…This cell model and similar I-Sce1 based system can be used for various purposes, some of which may include: comparing DNA repair via non homologous end-joining or homologous recombination [25,27] or their dependence on cell cycle [28,29]. I-Sce1 has also recently been used to develop a gene drive system in embryos to selectively eradicate malaria causing mosquito populations [34].…”

“…This system mimics the radiation dependent DNA damage that has previously been linked to bystander mutation in naïve cells. A similar cell line using I-Sce1 insert in intron 4 was constructed previously by other investigators to identify the relative contribution of DNA repair using the two dominant DNA repair pathways of non-homologous end-joining (NHEJ) and homologous recombination (HR) [27][28][29].…”