Rubio-Contreras, D. | Search bioRxiv

bioRxiv2026

The Holliday junction resolvase GEN1 preserves genome integrity and self-renewal in mouse embryonic stem cells

The maintenance of pluripotent stem cells (PSCs) under rapid proliferation requires mechanisms that both suppress replication-driven genome instability and preserve self-renewal capacity. Here, we show that, in contrast to somatic cells where it mainly acts as a backup, the Holliday junction resolvase GEN1 is required in mouse embryonic stem cells (ESCs), where its depletion severely compromises self-renewal and long-term maintenance. Loss of GEN1 induces the accumulation of cells with DNA content greater than 4C and chromosome fusions. Notably, a catalytically inactive GEN1 mutant rescues ESC colony formation, indicating that GEN1 supports ESC maintenance through non-enzymatic functions. In addition, GEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress and renders this phenotype dependent on DNA-PK activity, suggesting that GEN1 loss alters how pluripotent cells cope with replication-associated DNA lesions. Together, these findings identify GEN1 as a non-redundant guardian of genome integrity in pluripotent cells, revealing both a catalysis-independent role in self-renewal and a contribution to the replication stress response, with implications for PSC genomic quality control. HighlightsO_LIIn contrast to somatic cells, GEN1 is specifically required for mouse pluripotent cell self-renewal and expansion in vitro. C_LIO_LIGEN1 loss induces accumulation of DNA content greater than 4C and chromosome fusions without loss of core pluripotency markers expression. C_LIO_LICatalytically inactive GEN1 mutant rescues ESC colony-forming capacity. C_LIO_LIGEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress in a DNA-PK-dependent manner C_LI eTOCRamos-Lage et al. demonstrate that the resolvase GEN1 is essential for mouse embryonic stem cell self-renewal and genome stability. Strikingly, a catalytically dead mutant rescues colony formation, revealing an unexpected non-enzymatic role for GEN1 in pluripotency maintenance.

cell biology↗

bioRxiv2024

SCAN1 mutant TDP1 blocks the repair of DSB induced by TOP1 activity during gene transcription and promotes genome reorganisations and cell death in quiescent cells

DNA single-strand breaks (SSBs) are the most common type of DNA damage in quiescent cells, and defects in their repair can lead to hereditary neurological syndromes. A potential endogenous source of SSBs with pathogenic potential is the abortive activity of DNA topoisomerase 1 (TOP1) during transcription. Spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1), is caused by the homozygous mutation H493R in the gene encoding tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that initiates the repair of TOP1-induced SSBs by unlinking the TOP1 peptide from the break end. Notably, transcription-associated TOP1-induced SSBs can be converted into DNA double strand breaks (DSBs) in quiescent cells, with TDP1 also initiating the repair of these breaks. However, the role of TOP1-induced DSBs in the pathology of SCAN1 remains unclear. In this study, we have addressed the impact that SCAN1/H493R mutation, has in the repair of TOP1-induced DSB in quiescent cells. Here we demonstrate that while TDP1 deficiency delays the repair of these breaks, TDP1H493R completely blocks it. This blockage is accompanied by prolonged covalent trapping of TDP1H493R to DNA and results in genome instability and increased cell death in quiescent cells. We also demonstrate that tyrosyl-DNA phosphodiesterase 2 (TDP2) can backup TDP1 loss but not SCAN1 TDP1H493R mutation. Intriguingly, we also unveil that a mutation in catalytic H263 results in a negative dominant effect on TOP1-induced DSB repair. Collectively, our data provide novel insights into the molecular etiology of SCAN1 and support the potential of TOP1-induced DSBs as a main contributor to hereditary neurological syndromes.

genetics↗