Highlights d TOP2 binding and cleavage complex (TOP2cc) formation is dependent on cohesin d Transcription facilitates the processing of TOP2cc to DSBs that drives translocation d TOP2cc processing is rapid, while commitment to processing is rate-limiting d Cohesin and transcription levels predict TOP2-mediated breakage and translocation

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.

DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation ‘hotspot’, MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.

ObjectiveTo address the relationship between mutations in the DNA strand break repair protein tyrosyl DNA phosphodiesterase 2 (TDP2) and spinocerebellar ataxia autosomal recessive 23 (SCAR23) and to characterize the cellular phenotype of primary fibroblasts from this disease.MethodsWe have used exome sequencing, Sanger sequencing, gene editing and cell biology, biochemistry, and subcellular mitochondrial analyses for this study.ResultsWe have identified a patient in the United States with SCAR23 harboring the same homozygous TDP2 mutation as previously reported in 3 Irish siblings (c.425+1G>A). The current and Irish patients share the same disease haplotype, but the current patient lacks a homozygous variant present in the Irish siblings in the closely linked gene ZNF193, eliminating this as a contributor to the disease. The current patient also displays symptoms consistent with mitochondrial dysfunction, although levels of mitochondrial function in patient primary skin fibroblasts are normal. However, we demonstrate an inability in patient primary fibroblasts to rapidly repair topoisomerase-induced DNA double-strand breaks (DSBs) in the nucleus and profound hypersensitivity to this type of DNA damage.ConclusionsThese data confirm the TDP2 mutation as causative for SCAR23 and highlight the link between defects in nuclear DNA DSB repair, developmental delay, epilepsy, and ataxia.