Abstract
Our genomes harbor conserved DNA sequences, known as common fragile sites (CFSs), that are difficult to replicate and correspond to regions of genome instability. Following replication stress, CFS loci give rise to breaks or gaps (termed CFS expression) where under-replicated DNA subsequently undergoes mitotic DNA synthesis (MiDAS). We show that loss of the structure-selective endonuclease GEN1 reduces CFS expression, leading to defects in MiDAS, ultrafine anaphase bridge formation, and DNA damage in the ensuing cell cycle due to aberrant chromosome segregation. GEN1 knockout cells also exhibit an elevated frequency of bichromatid constrictions consistent with the presence of unresolved regions of under-replicated DNA. Previously, the role of GEN1 was thought to be restricted to the nucleolytic resolution of recombination intermediates. However, its ability to cleave under-replicated DNA at CFS loci indicates that GEN1 plays a dual role resolving both DNA replication and recombination intermediates before chromosome segregation.