Wang LHC, Schwarzbraun T, Speicher MR, Nigg EA.. Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation. Chromosoma 117: 123-135

Department of Cell Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany.
Chromosoma (Impact Factor: 4.6). 05/2008; 117(2):123-35. DOI: 10.1007/s00412-007-0131-7
Source: PubMed


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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    • "Taken together with the herepresented in vitro analysis, this could suggest that Smc5/6 facilitates Top2- dependent formation of SCIs. However, reduction of the function of either Smc5/ 6 or Top2 leads to the formation of unresolved DNA bridges at anaphase (Gallego-Paez et al., 2014; Rouzeau et al., 2012; Wang et al., 2008), indicating a function for Smc5/6 in the resolution of SCIs. Accordingly, Smc5/6 accumulates on S. cerevisiae chromosomes after inhibition of Top2 (Jeppsson et al., 2014; Kegel et al., 2011). "
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    ABSTRACT: The structural maintenance of chromosome (SMC) protein complexes cohesin and condensin and the Smc5/6 complex (Smc5/6) are crucial for chromosome dynamics and stability. All contain essential ATPase domains, and cohesin and condensin interact with chromosomes through topological entrapment of DNA. However, how Smc5/6 binds DNA and chromosomes has remained largely unknown. Here, we show that purified Smc5/6 binds DNA through a mechanism that requires ATP hydrolysis by the complex and circular DNA to be established. This also promotes topoisomerase 2-dependent catenation of plasmids, suggesting that Smc5/6 interconnects two DNA molecules using ATP-regulated topological entrapment of DNA, similar to cohesin. We also show that a complex containing an Smc6 mutant that is defective in ATP binding fails to interact with DNA and chromosomes and leads to cell death with concomitant accumulation of DNA damage when overexpressed. Taken together, these results indicate that Smc5/6 executes its cellular functions through ATP-regulated intermolecular DNA linking. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "In animal cells, chromosomes are typically longer than in budding yeast, and inactivation of topo II as late as anaphase leads to chromosome missegregation, indicating that SCI persist until late cell cycle stages (Ishida et al., 1994; Oliveira et al., 2010). Consistently, anaphase DNA bridges that are resolved in a topo II–dependent manner have been observed in human cells (Baumann et al., 2007; Chan et al., 2007; Wang et al., 2008). Thus, whether there are differences in the time of SCI resolution between budding yeast and animal cells and, if so, what the underlying reasons are remain unclear. "
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    ABSTRACT: To allow chromosome segregation, topoisomerase II (topo II) must resolve sister chromatid intertwines (SCI) formed during deoxynucleic acid (DNA) replication. How this process extends to the full genome is not well understood. In budding yeast, the unique structure of the ribosomal DNA (rDNA) array is thought to cause late SCI resolution of this genomic region during anaphase. In this paper, we show that chromosome length, and not the presence of rDNA repeats, is the critical feature determining the time of topo II-dependent segregation. Segregation of chromosomes lacking rDNA also requires the function of topo II in anaphase, and increasing chromosome length aggravates missegregation in topo II mutant cells. Furthermore, anaphase Stu2-dependent microtubule dynamics are critical for separation of long chromosomes. Finally, defects caused by topo II or Stu2 impairment depend on attachment of telomeres to the nuclear envelope. We propose that topological constraints imposed by chromosome length and perinuclear attachment determine the amount of SCI that topo II and dynamic microtubules resolve during anaphase.
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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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