RESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Mutação , Doenças Neurodegenerativas , Diester Fosfórico Hidrolases , Humanos , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Mutação/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , Transcrição Gênica , Estruturas R-Loop , Sistemas CRISPR-Cas/genéticaRESUMO
Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) is limited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.
