Active, but not inactive, human centromeres display topoisomerase II activity in vivo.
Eukaryotic centromeres are composed of centromere DNA and the multiple proteins directly or indirectly associated with it. One important DNA-binding protein in the centromere is DNA topoisomerase II (topo II). In the genome in general, topo II has two functions, one structural and one enzymatic, the latter catalyzing DNA strand-passage reactions. It has been demonstrated that topo II accumulates at centromeres during the first part of mitosis, and disappears again at anaphase, but it has not been clear whether it serves a structural or an enzymatic function at the centromere. To investigate this issue, we developed the topo II-induced self-primed in situ assay (Topo-SPRINS). In this assay, DNA breaks created by topo II are stabilized with the topo II inhibitor VM-26 in vivo, and used as 'primers' for localized DNA synthesis in vitro. The assay revealed that topo II has enzymatic activity at mitotic centromeres and that the activity is relatively constant across centromeres. Furthermore, the activity was observed at a neocentromere, and, in multicentric chromosomes, the activity was restricted to the active centromere. The topo II activity is thus selectively present at functioning centromeres, indicating that it plays a role in mitotic centromere function.
Cancercytogenetics Laboratory, Tage Hansens Gade 2, DK-8000 Aarhus C, Denmark.
Claus L
CL
Andersen
Annelise
A
Wandall
Eigil
E
Kjeldsen
Christian
C
Mielke
Jørn
J
Koch
D002503:Centromere / Q000201:enzymology*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D013713:Teniposide; D004250:DNA Topoisomerases, Type II