Tamara Eleanore Hamann, Angela Wieland, Farbod Mohensi, Kruno Vukušić, Andrea Tirincsi, Rene Wardenaar, Marialucrezia Losito, Iris Harmsen, Ipek Ilgin Gönenc, Bernd Wollnik, Floris Foijer, Iva M. Tolić, Zuzana Storchová, Markus Räschle

• The BLM helicase is a critical genome maintenance protein in v olv ed in div erse cellular processes including DNA replication, repair, transcription, and chromosome segregation. During mitosis, it cooperates with the PICH helicase and topoisomerases to resolve ultrafine DNA bridges (UFBs)—nonchromatinized DNA str uct ures that link sister chromatids—through a mechanism that is not yet fully understood. Here, we tagged endogenous BLM and PICH with fluorescent proteins and BLM with an auxin-inducible degron to generate a cell model system that enables temporal tracking of UFB dynamics in the presence or absence of BLM. Time-resolved lattice light sheet microscopy established the dynamic localization patterns of BLM and PICH throughout the cell cy cle. While BLM cy cles betw een PML bodies and DNA repair foci in interphase, these str uct ures disappear at the mitotic entry, and BLM then re-associates with chromatin during anaphase to UFBs as well as to CENP-B- positive mitotic foci. Acute BLM depletion during mitosis increased the fraction of unresolved UFBs, micronuclei containing acentric fragments, binucleation, and resulted in subtle genomic abnormalities detected by single-cell whole genome sequencing. These findings highlight a mitosis- specific role for BLM in UFB resolution and underscore its function in preserving genomic stability.