MAD2B guides chromosome stability in mitosis via the MAD2B–CIP2A signaling axis

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, monitored by the spindle assembly checkpoint machinery, which prevents precocious chromosome segregation and reduces the risk of aneuploidy. MAD2B, a homologue of MAD2, is critical for mitotic quality control and DNA damage repair. However, it remains elusive how MAD2B guides genome surveillance during the cell cycle. Here, we show that MAD2B safeguards genome integrity through interactions with CIP2A in a context-dependent manner. To delineate the molecular mechanisms underlying MAD2B-dependent signaling in mitosis, we carried out affinity purification of FLAG-MAD2B followed by mass spectrometry to identify MAD2B-associated proteins. Our biochemical characterization uncovered a previously uncharacterized interaction between MAD2B and CIP2A, mediated by the N-terminus of CIP2A. Importantly, MAD2B depletion resulted in DNA damage response and replication stress phenotypes, leading to aberrant mitotic DNA synthesis primarily in HeLa cells. Notably, loss of MAD2B disrupted CIP2A recruitment to γH2AX-marked DNA lesions and attenuated DNA damage repair. Together, these results establish MAD2B as a context-sensitive regulator of genome stability that links replication stress surveillance to mitotic chromosome repair via the MAD2B-CIP2A signaling axis.