Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells.

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named ‘centromere disintegration’ that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.

Running title: PLK1 protects centromere integrity during mitosis Key words: PLK1, PICH/ERCC6L, BLM, ultrafine DNA bridge, PIT, chromosome biorientation, centromere dislocation, metaphase collapse collapse'. In contrast, cells treated with the APC/C inhibitor, ProTAME, remained at metaphase for extended periods . More strikingly, we found that during 'metaphase collapse', cells produced a thread-like structure that was decorated with the PLK1 protein, which was not detected in untreated mitotic cells ( Fig. 1d; arrows). Since the PLK1-coated threads were reminiscent of ultrafine DNA bridges (UFBs) that we and others identified previously in anaphase cells 7, 8 , we investigated whether they are DNA molecules; or alternatively a mis-localisation of PLK1 to cytoskeleton structures. We found that both well-known UFB-binding factors, PICH translocase and BLM helicase were present along the PLK1-coated thread structures (Figs. 1e, f and Supplementary Fig. 1d). Moreover, there was also a strong staining of replication protein A (RPA), indicating the presence of single-stranded DNA (Fig. 1f). The immunostaining results were further confirmed by examining Bloom's syndrome fibroblast cells stably expressing a GFPtagged BLM and RPE1 cells expressing a GFP-tagged PLK1 (Supplementary Figs. 1e, 1f; arrows). These data indicate that the PLK1-coated thread structures formed during 'metaphase collapse' are DNA molecules. Consistent with this, we also found that PLK1 is indeed a component of UFB-binding complex and localises to UFBs in normal anaphase cells ( Supplementary Fig. 1g; arrows). As predicted, the DNA threads did not co-localise with mitotic microtubules ( Supplementary Fig. 1h). Intriguingly, all DNA threads analysed (n=171) showed either one or both of their termini exclusively originating from centromeric regions (Fig. 1g). In some optical sections, it was obvious that two separating centromeres were interlinked by a DNA thread molecule ( Fig. 1g; arrows). Alongside RPE1 cells, a short treatment of BI2536 in other normal human cells (including primary cells) and cancer cells also induced metaphase misalignment and centromeric DNA threads, albeit with different frequencies ( Supplementary Fig. 2a; 82-6 (24%), 1BR3 (31%) and HCT116 (69%)). A plausible explanation for the formation of centromeric DNA threads could be due to precocious loss of sister chromatid cohesion, similar to the effect of SGO1 depletion 17,18 , which leads to the exposure of sister DNA catenanes at centromeres 8 ( Supplementary Fig. 2b; arrows). However, consistent with published studies 19-21 , inhibition of PLK1 did not induce premature sister-chromatid separation in RPE1 cells ( Supplementary Fig. 2c). Most crucially, by co-staining with the chromatid axis marker, TOP2A, we clearly visualised that chromosomes, which comprised of a pair of cohesed sister chromatids, were connected by DNA threads at their centromeres ( Fig. 1h; arrows). Therefore, the loss of PLK1 function simultaneously induces collapse of metaphase and inter-chromosomal DNA ...