Hairpin structures formed by alpha satellite DNA of human centromeres are cleaved by human topoisomerase IIalpha

Abstract

Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIalpha-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIalpha-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion.

Figure 1.

Topoisomerase IIα introduces a single-stranded break at the loop of a hairpin structure formed by the conserved part of centromeric alpha satellite DNA. (A) Topoisomerase IIα-mediated cleavage of the 3′-end labelled substrates, TOP83 (lanes 2–6), TOP83 hybridized to BOT83 (lanes 8–12), or BOT83 (lanes 14–18). The asterisk indicates radioactive labelling of the substrate. The incubation time for the cleavage reaction is indicated above the individual lanes. Lanes 1 and 13, DNA marker increasing in steps of 10 bases; lanes 2, 8 and 14, DNA controls without topoisomerase IIα; lane 7, digestion of the labelled duplex substrate with the restriction enzyme DdeI. Migration of the cleavage fragments is retarded with one nucleotide due to a small peptide remaining covalently linked to the DNA after proteinase K treatment. The smear above the cleavage bands represents cleavage products having a longer protein fragment covalently linked due to partial proteinase K digestion (37). (B) Predicted secondary structure of TOP83, the major and minor cleavage sites are indicated by a thick and a thin arrow, respectively. The radioactive nucleotide added to the substrate in the labelling reaction is indicated by a bold ‘A’ with an asterisk.

Figure 2.

Topoisomerase IIα-mediated cleavage of the centromeric hairpin is differentially affected by VM26 and mAMSA. (A) Topoisomerase IIα-mediated cleavage of TOP83 in the absence or presence of the indicated concentrations of VM26 and mAMSA. Experiments with DMSO were used as reference, as VM26 and mAMSA were dissolved in DMSO. ‘Standard’ denotes experiments without DMSO and drugs. The results are the means +/– SD of four independent experiments. (B) Topoisomerase IIα-mediated cleavage of TOP83 in the absence or presence of the indicated concentrations of ethidium bromide (EtBr). Cleavage experiments were performed as described in (A). Cleavage levels are relative to cleavage levels obtained in the absence of EtBr. The results are the means +/– SD of two independent experiments.

Figure 3.

Topoisomerase IIα-mediated cleavage of the centromeric hairpin is determined by an unpaired base in the stem of the hairpin structure. (A) Hairpin structures of the oligonucleotides TOP62 and BOT62, which are identical to TOP83 and BOT83, respectively, except for the lack of most of symmetry 2. The arrow indicates the position of topoisomerase IIα-mediated cleavage; open arrowheads represent positions involved in the mutational analysis shown in (B). The radioactive nucleotide added to the substrate in the labelling reaction is indicated by a bold ‘A’ with an asterisk. (B) Topoisomerase IIα-mediated cleavage of mutated forms of TOP62 and BOT62. The incubation time was 90 min in all cleavage experiments. Migration of the cleavage fragments is retarded with one nucleotide due to a small peptide remaining covalently linked to the DNA after proteinase K treatment. The smear above the cleavage bands represents cleavage products having a longer protein fragment covalently linked due to partial proteinase K digestion (37). The specific mutations are indicated above the lanes. Lane 1, DNA marker increasing in steps of 2 bases; lane 11, DNA marker increasing in steps of 10 bases.

Figure 4.

Topoisomerase IIα-mediated cleavage of the centromeric hairpin is inhibited if the distance between the unpaired base and the hairpin loop is increased. (A) Topoisomerase IIα-mediated cleavage of TOP62 derived substrates having 1, 2, 3 or 4 extra base pairs inserted between the unpaired base and the hairpin loop as indicated above the lanes. Lanes 1 and 7, DNA marker increasing in steps of 10 bases. The asterisks indicate cleavage products in lane 6, which migrate slower due to partial proteinase K digestion. The substrates are schematically presented in (B). The arrows indicate the cleavage site.

Figure 5.

‘Kissing-loop’ model of two centromeric DNA hairpins bound to topoisomerase IIα. (A) Two DNA hairpins located loop-to-loop will fulfil the requirement of the enzyme for binding ∼26 bp of DNA. The ‘kissing-loops’ mimic a standard DNA duplex allowing human topoisomerase IIα to introduce a ‘double-stranded break’ as indicated by the arrows. (B) Simultaneous topoisomerase IIα-mediated cleavage of two ‘top strand’ hairpins may provide cohesion of sister-chromatids in the centromeric region. Since the enzyme only binds top strand hairpins, the two hairpins held together and cleaved by topoisomerase IIα must originate from two different alpha satellite monomers. If the two monomers belong to two-sister chromatids, the enzyme will bind the sister-chromatids together until DNA religation has taken place.