Site-Specific Cleavage by Topoisomerase 2: A Mark of the Core Centromere.

In addition to its roles in transcription and replication, topoisomerase 2 (topo 2) is crucial in shaping mitotic chromosomes and in ensuring the orderly separation of sister chromatids. As well as its recruitment throughout the length of the mitotic chromosome, topo 2 accumulates at the primary constriction. Here, following cohesin release, the enzymatic activity of topo 2 acts to remove residual sister catenations. Intriguingly, topo 2 does not bind and cleave all sites in the genome equally; one preferred site of cleavage is within the core centromere. Discrete topo 2-centromeric cleavage sites have been identified in α-satellite DNA arrays of active human centromeres and in the centromere regions of some protozoans. In this study, we show that topo 2 cleavage sites are also a feature of the centromere in Schizosaccharomyces pombe, the metazoan Drosophila melanogaster and in another vertebrate species, Gallus gallus (chicken). In vertebrates, we show that this site-specific cleavage is diminished by depletion of CENP-I, an essential constitutive centromere protein. The presence, within the core centromere of a wide range of eukaryotes, of precise sites hypersensitive to topo 2 cleavage suggests that these mark a fundamental and conserved aspect of this functional domain, such as a non-canonical secondary structure.

Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.

Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells.

DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

Depletion of topoisomerase IIalpha leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads.

Topoisomerase II (topo II) is a major component of mitotic chromosomes, and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/molecular mapping of the catalytic activity of topo II to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. By using a human cell line with a conditional-lethal mutation in the gene encoding DNA topoisomerase IIalpha, we find that depletion of topo IIalpha, while leading to a disorganised metaphase plate, does not have any overt effect on general assembly of kinetochores. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to depletion of topo IIalpha might be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres and the abnormal persistence of PICH-coated connections between segregating chromatids.

Increased missegregation and chromosome loss with decreasing chromosome size in vertebrate cells.

Chromosome engineering has allowed the generation of an extensive and well-defined series of linear human X centromere-based minichromosomes, which has been used to investigate the influence of size and structure on chromosome segregation in vertebrate cells. A clear relationship between overall chromosome size and mitotic stability was detected, with decreasing size associated with increasing loss rates. In chicken DT40, the lower size limit for prolonged mitotic stability is approximately 550 kb: at 450 kb, there was a dramatic increase in chromosome loss, while structures of approximately 200 kb could not be recovered. In human HT1080 cells, the size threshold for mitotic stability is approximately 1.6 Mb. Minichromosomes of 0.55-1.0 Mb can be recovered, but display high loss rates. However, all minichromosomes examined exhibited more segregation errors than normal chromosomes in HT1080 cells. This error rate increases with decreased size and correlates with reduced levels of CENP-A and Aurora B. In mouse LA-9 and Indian muntjac FM7 cells, the size requirements for mitotic stability are much greater. In mouse, a human 2.7-Mb minichromosome is rarely able to propagate a kinetochore and behaves acentrically. In Indian muntjac, CENP-C associates with the human minichromosome, but the mitotic apparatus appears unable to handle its segregation.

Topoisomerase II cleavage activity within the human D11Z1 and DXZ1 alpha-satellite arrays.

Topoisomerase II (Topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of Topo II, most probably because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached principally through their centromeric regions. Intriguingly, evidence has recently been presented for Topo II cleavage activity within the centromeric alpha-satellite DNA arrays of the human X and Y chromosomes. In this report we extend these observations by mapping distinct sites of Topo II cleavage activity within the alpha-satellite array of human chromosome 11. A single major site of cleavage has been assigned within the centromeric DNA of each of three independently derived, and active, 11 centromeres. Unlike the X and Y centromeres, where cleavage sites mapped close to (within 150 kb of) the short arm edge of the arrays, on chromosome 11, the cleavage sites lie many hundreds of kilobases into each alpha-satellite array. We also demonstrate that catalytically active Topo II is concentrated within the centromere domain through an extended period of G2 and M, with levels declining in G1 and S.

Co-localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X alpha-satellite array.

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIalpha cleavage activity; and mitotic stability. We have determined that the human X major alpha-satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored approximately 150 kb in from the Xp-edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.