Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation

Abstract

PICH (Plk1-interacting checkpoint helicase) was recently identified as an essential component of the spindle assembly checkpoint and shown to localize to kinetochores, inner centromeres, and thin threads connecting separating chromosomes even during anaphase. In this paper, we have used immuno-fiber fluorescence in situ hybridization and chromatin-immunoprecipitation to demonstrate that PICH associates with centromeric chromatin during anaphase. Furthermore, by careful analysis of PICH-positive anaphase threads through FISH as well as bromo-deoxyurdine and CREST labeling, we strengthen the evidence that these threads comprise mainly alphoid centromere deoxyribonucleic acid. Finally, by timing the addition of ICRF-193 (a specific inhibitor of topoisomerase-II alpha) to cells synchronized in anaphase, we demonstrate that topoisomerase activity is required specifically to resolve PICH-positive threads during anaphase (as opposed to being required to prevent the formation of such threads during earlier cell cycle stages). These data indicate that PICH associates with centromeres during anaphase and that most PICH-positive threads evolve from inner centromeres as these stretch in response to tension. Moreover, they show that topoisomerase activity is required during anaphase for the resolution of PICH-positive threads, implying that the complete separation of sister chromatids occurs later than previously assumed.

Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division

“…However, the discovery of PICH-positive anaphase threads strongly suggests the existence of a transient physical connection between the centromeres of sister chromatids even after checkpoint silencing and anaphase onset (Baumann et al, 2007;Wang et al, 2008). Thus, the functional relationship between the resolution of cohesin-mediated cohesion and DNA catenation remains to be fully understood, particularly for chromosomes of higher eukaryotes (Diaz-Martinez et al, 2007).…”

“…In human cells, the decatenation of DNA double strands is brought about primarily by Topoisomerase-II (Topo-II; also known as DNA Topoisomerase-II) and most catenation along chromosome arms is resolved before metaphase (Porter and Farr, 2004). At centromeres, however, the resolution of catenated DNA appears to be completed only during anaphase, as inferred from recent studies demonstrating the persistence of PICH (Plk1-interacting checkpoint helicase; also known as DNA excision repair protein ERCC-6 like)-positive DNA threads upon inhibition of Topo-II in human anaphase cells (Baumann et al, 2007;Wang et al, 2008). Similarly, a requirement for Topo-II activity after anaphase onset has been shown in a number of different organisms (Downes et al, 1991;Gimenez-Abian et al, 2002;Shamu and Murray, 1992;Uemura et al, 1987).…”

“…Moreover, a subpopulation of UFBs was recently shown to connect fragile site loci associated with Fanconi anaemia proteins FANCD2 and FANCI, thus, probably representing abnormal intertwined DNA structures induced by replicative stress (Chan et al, 2009;Naim and Rosselli, 2009). However, considering that a majority of PICH-positive UFBs connects the kinetochores of separating sister chromatids even in unperturbed cells (Baumann et al, 2007;Wang et al, 2008), it seems plausible that these represent physiological rather than pathophysiological structures.…”

The perpetual movements of anaphase

“…It has been suggested that chromosome velocity is slow to allow the decatenation of entangled DNA strands on the sister chromatids through the action of Topoisomerase II [12]. The recent discovery of PICH, a protein that associates with persistent DNA threads during anaphase, whose resolution is dependent on the activity of Topoisomerase II, strongly supports this view [13,14]. The slow movement of chromosomes during anaphase may also provide the necessary time to correct and accurately segregate chromosomes with merotelic attachments, i.e., chromosomes with individual kinetochores bound to microtubules from both poles [15,16].…”

A new class of ultrafine anaphase bridges generated by homologous recombination

“…CENP-A, HEC1) at the bridges’ termini. C-UFBs exist in every mitosis and their numbers are increased by treatment with the topoisomerase IIα inhibitor ICRF-193, indicating that they are readily removed by this topoisomerase [5,6,9,10]. Second, DNA catenanes that persist at ribosomal DNA (rDNA) loci give rise to R-UFBs that colocalise with the ribosomal RNA transcription factor UBF, a marker for rDNA [11].…”