Abstract
<jats:p>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.</jats:p>