TI - DISCUSSION . AB - It is well established that the C-terminal domains of both yeast and human topoisomerase II are dispensable for the enzyme's basic catalytic activity ( 55,61 ) . On the other hand , a large body of evidence suggests that the C-terminal domain plays a role in regulating the cellular functioning of topoisomerase II . Most notably , it contains crucial nuclear localization signals ( 55,62,63 ) and sites PHOSphorylated in a cell-cycle-related manner ( 38 ) . Since the C-terminal domains are the most divergent portions of the two mammalian isoforms of topoisomerase II ( 16 ) , it has been proposed that they determine specific functions differing between these two isoforms in vivo ( 39 ) . Here , we provide direct evidence in support of this view . We demonstrate that the divergent CTRs of topoisomerase IIalpha and IIbeta govern two features in which the two isoforms characteristically differ , namely binding to mitotic chromosomes and support of cell proliferation . We show that YFP-fused topoisomerase IIalpha is preferentially chromosome -bound during mitosis and fully supports proliferation of cells lacking endogenous topoisomerase IIalpha . In contrast , the majority of YFP-fused topoisomerase IIbeta is not chromosome -bound at mitosis and clones emerged from complementation experiments at greatly reduced frequencies . The specific features of the alpha isoform were stringently linked to the presence of the alpha CTR . Replacement of the CTR in topoisomerase IIbeta with the alpha CTR produced an enzyme chimera that behaved like topoisomerase IIalpha , whereas the converse experiment produced an enzyme chimera behaving like topoisomerase IIbeta . The requirement of topoisomerase IIalpha for proper cell division has been suggested by indirect evidence showing an essential role in chromosome segregation , which is not readily adopted by the beta-isoform ( eg 25 ) . More recently , depletion of topoisomerase IIalpha by various experimental strategies resulted in each case in an impaired separation of chromosomes in anaphase. ( 26,27,64 ) . Here we describe a striking coincidence between the ability of all versions of topoisomerase II furnished with the alpha CTR to complement such a lack of endogenous topoisomerase IIalpha and the propensity of the complementing construct to bind to mitotic chromosomes . We even observe that the alpha CTR alone preferentially binds to metaphase chromosomes . It remains unclear whether chromosome binding is due to direct DNA-interactions , as suggested for various prokaryotic type II topoisomerases ( 65,66 ) , or to interactions with other proteins , e.g.condensins ( 67 ) or HSP90 ( 68 ) . However , the strict correlation between the binding to metaphase chromosomes and the support of cell proliferation suggests a mechanistic connection between the two . It can be hypothesized (i) that efficient separation of sister chromatids and proper cell division depend on a high , local concentration of active topoisomerase II at the mitotic chromosome , ( ii ) that under physiological conditions only the alpha isoform accumulates in sufficient concentrations at the mitotic chromosome , and ( iii ) that this feature is promoted by an intrinsic ability of the alpha CTR to bind to metaphase chromosomes . These hypotheses would assign to the alpha CTR the function of an adaptor that shifts the binding equilibrium of the entire enzyme molecule towards the bound state and thus provides the chromosome at mitosis with sufficient topoisomerase II activity to perform extensive DNA-decatenation in the course of sister chromatid segregation . Our finding that topoisomerase IIbeta can also support cell proliferation when expressed at extremely high levels supports such a hypothesis : sufficient local concentration of active topoisomerase II at the mitotic chromosome cannot only be acquired by expressing normal levels of an enzyme having a high affinity ( due to the alpha CTR ) , but also by expressing highly increased levels of an enzyme having a much lower one ( due to the beta CTR ) . The above interpretation is more difficult to fit with complementation studies carried out in yeast that show that both mammalian isoforms are equally capable of rescuing temperature-sensitive Deltatop2 yeast mutants ( 22,23 ) . One explanation could be that yeast might be unable to discriminate between the two mammalian isoforms . However , this explanation is unlikely because mouse topoisomerase IIalpha and IIbeta can be discriminated by yeast , in as much as they are distributed in a distinguishable manner in yeast cell nuclei ( 57 ) . Another explanation could be that yeast is more tolerant to changes in topoisomerase II expression levels than human cells , which are readily killed by overexpression of these enzymes ( 69 ) . Since high copy number vectors were used in the yeast studies for expression of the complementing enzymes , expression levels of the beta isoform could have been high enough to enable efficient complementation of topoisomerase II functions in the same manner as seen here in a human cell line . Another possible interpretation of our data is that the topoisomerase IIalpha plays an essential role during replication . It has been shown in yeast that topoisomerase II is required for DNA replication , when topoisomerase I is lacking ( 70 ) , because movement of DNA replication complexes through the DNA double helix induces positive supercoils ahead of this machinery . Recent work in yeast demonstrates that topoisomerase II relaxes chromatin even more efficiently than topoisomerase I ( 71 ) . In mammals , topoisomerase IIalpha appears to be a key player in removal of this type of torsional stress during replication ( 17 ) , and it was postulated that this isoform-specificity is determined by the divergent C-terminal regions ( 72 ) . The residues that were suggested to play this role in replication are all within the alpha CTRs analyzed here . In addition , a study in chicken fibroblast showed that topoisomerase IIalpha co-localizes with sites of replication , and this targeting was also mediated by the alpha CTR ( 73 ) . Unfortunately , it is difficult to determine whether topoisomerase IIalpha plays a truly essential role during replication that cannot be complemented by other proteins ( eg topoisomerase I or topoisomerase IIbeta ) . Although silencing of topoisomerase IIalpha in human cells ( 26 ) and mice ( 27 ) causes a defect in chromosome segregation , suggesting that its essential role is during mitosis rather than S-phase , this phenotype could conceivably be caused by loss of an essential topoisomerase IIalpha function during the late phases of replication . Lack of such a function could still allow for a progression into metaphase followed by mitotic catastrophes due to unresolved DNA catenanes . Thus , an essential role of topoisomerase IIalpha in relaxation of positive supercoils generated at late stages of replication cannot be excluded , and our observation that the alpha CTR is required for efficient support of cell proliferation by topoisomerase II may just as well reflect a specific involvement of the alpha isoform in DNA replication ( 72 ) . Regardless of the exact role of the alpha CTR , we demonstrate in this article that it confers a unique , proliferation-associated functionality to the topoisomerase II core enzyme ( either version of it ) , whereas the beta CTR is much less efficient in this respect . It is therefore plausible that the two versions of the CTR cooperate in differential targeting of topoisomerase IIalpha and IIbeta , thus providing unique functionality of the two isoforms in proliferating cells .