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

Department of Cell Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany.
Chromosoma (Impact Factor: 3.26). 05/2008; 117(2):123-35. DOI: 10.1007/s00412-007-0131-7
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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.

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Available from: Thomas Schwarzbraun, Aug 18, 2015
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    • "Since it has been reported that the helicase PICH appears at centromeric ultrafine anaphase bridges in somatic cultured cells (Baumann et al. 2007; Spence et al. 2007; Wang et al. 2008; Ke et al. 2011; Kaulich et al. 2012), we studied the relative distribution of PICH and Topo IIα during the metaphase II/anaphase II transition to assess whether they colocalised at TOCOSs. By using an antibody against human PICH, we were not able to detect centromere signals during the second meiotic division. "
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    ABSTRACT: Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.
    Chromosoma 09/2013; 123(1-2). DOI:10.1007/s00412-013-0434-9 · 3.26 Impact Factor
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    • "According to one interesting recent proposal, PICH cooperates with the BLM helicase to unravel chromatin, thereby creating nucleosome-free areas, so that catenated or aberrant DNA structures can be resolved (Ke et al. 2011). In other studies, the possibility has been emphasized that PICH may act as a chromatin remodeling enzyme to facilitate the action of topoisomerase II (Wang et al. 2010, 2008). Whatever the precise role of PICH, our current results clearly indicate that this function requires its ATPase activity. "
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    ABSTRACT: The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation. Electronic supplementary material The online version of this article (doi:10.1007/s00412-012-0370-0) contains supplementary material, which is available to authorized users.
    Chromosoma 04/2012; 121(4):395-408. DOI:10.1007/s00412-012-0370-0 · 3.26 Impact Factor
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    • "When cells are directly fixed without manipulations, these threads are devoid of histones, cannot be stained with DNA intercalating dyes such as DAPI (4 0 ,6-diamidino-2-phenylindole), and frequently connect separated centromere pairs (Baumann et al, 2007; Wang et al, 2008). Because inactivation of topoisomerase IIa (Topo IIa) increases the frequency and persistence of PICH-positive threads in cells, it is highly likely that PICH can decorate catenated centromeric DNA (Spence et al, 2007; Wang et al, 2008). PICH was also proposed to function in the spindle checkpoint (hence its name) and to regulate the kinetochore localization of Mad2, a key spindle checkpoint protein (Baumann et al, 2007). "
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    ABSTRACT: Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.
    The EMBO Journal 08/2011; 30(16):3309-21. DOI:10.1038/emboj.2011.226 · 10.75 Impact Factor
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