ABSTRACT
TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Mutation , Neurodegenerative Diseases , Phosphoric Diester Hydrolases , Humans , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/genetics , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Mutation/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type I/genetics , Transcription, Genetic , R-Loop Structures , CRISPR-Cas Systems/geneticsABSTRACT
Transcription is an essential cellular process but also a major threat to genome integrity. Transcription-associated DNA breaks are particularly detrimental as their defective repair can induce gene mutations and oncogenic chromosomal translocations, which are hallmarks of cancer. The past few years have revealed that transcriptional breaks mainly originate from DNA topological problems generated by the transcribing RNA polymerases. Defective removal of transcription-induced DNA torsional stress impacts on transcription itself and promotes secondary DNA structures, such as R-loops, which can induce DNA breaks and genome instability. Paradoxically, as they relax DNA during transcription, topoisomerase enzymes introduce DNA breaks that can also endanger genome integrity. Stabilization of topoisomerases on chromatin by various anticancer drugs or by DNA alterations, can interfere with transcription machinery and cause permanent DNA breaks and R-loops. Here, we review the role of transcription in mediating DNA breaks, and discuss how deregulation of topoisomerase activity can impact on transcription and DNA break formation, and its connection with cancer.
