L. de Haan, R. Schneeweiss, M. Barazas, M. L. Kaptein, F. J. Bakker, A. Dekker, E. Ovcharenko, S. S. Beneddine, A. Graf, D. Paouneskou, B. van de Kooij, R. Freire, V. Jantsch, J. Jonkers, H. R. de Boer, M. Tijsterman, M. A. T. M. van Vugt, P. J. Huis in 't Veld
DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.