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

Abstract

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.

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks

“…Human TOP2A recognizes and cleaves within the single-stranded DNA loop region of the hairpin structure formed within alpha satellite DNA ( 36 ), and human TOP2B binds and cleaves four-way junction DNA structure ( 34 ). Site-specific cleavage by TOP2 at centromeric DNA with dyad symmetries (potential to form hairpins and four-way junctions) is found in yeast, fruit fly, chicken and human ( 35 , 37 ). Moreover, mismatched bases which are often present in multiple stem-loop structures, when in the proximity of TOP2 cleavage sites can greatly stimulate TOP2 cleavage activity and hinder DNA end re-ligation ( 38 , 39 ).…”

Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands

“…Interestingly, recent studies demonstrate a major contribution of TOP2 activity in the generation of DSBs in highly transcribed genes ( Canela et al, 2019 ; Gittens et al, 2019 ; Gothe et al, 2019 ). Moreover, several studies demonstrated that DNA topoisomerase 1 (TOP1) and TOP2 recognize and preferentially cleave DNA at regions forming stable secondary structures ( Froelich-Ammon et al, 1994 ; Jonstrup et al, 2008 ; Mills et al, 2018 ; West and Austin, 1999 ). In eukaryotic cells, TOP1 and TOP2 activities are required to resolve topological stresses resulting from DNA transactions (for a review, see Pommier et al, 2016 ).…”

Broken by the Cut: A Journey into the Role of Topoisomerase II in DNA Fragility

“…TOP2 responds to torsional stress generated in the maintenance of the genome; however, supercoiling is not required and TOP2 does not have a stringent recognition sequence [67,68]. Interestingly, there is evidence that alternative DNA secondary structures are targeted by topoisomerase for enzymatic cleavage (Figure 2A) [69,70,71,72]. Specifically, Froelich-Ammon et al demonstrated that TOP2 can recognize and cleave stem-loop structures one nucleotide from the 3′ base of the stem structure [69].…”

“…Additionally, TOP2 was shown to cleave the single-stranded loop of stem-loop structures formed in alpha-satellite sequences. This activity is seen in the alpha-satellite contained in centromeres across multiple species, which further points to a non-B DNA secondary structure as the common site for TOP2 cleavage [70,71]. Furthermore, West and Austin demonstrated that TOP2B not only binds and cleaves four-way junction DNA, but also has on average a four-fold higher affinity for four-way junction DNA compared to linear DNA [72].…”

“…Interestingly, chemical-induced breaks within this region were mapped to predicted TOP1 and TOP2 cleavage sites on these secondary structures. As topoisomerase enzymatic activity is enhanced by DNA secondary structures, this suggests that the fragility associated with RET intron 11 is due to the activity of topoisomerases at the DNA secondary structures [13,69,70,71]. Future research remains to be done to understand the role of TOP2 in facilitating oncogenic rearrangements in both hematological and solid cancers.…”