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DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.

During mitosis, from late prophase onward, sister chromatids are connected along their entire lengths by axis-linking chromatin/structure bridges. During prometaphase/metaphase, these bridges ensure that sister chromatids retain a parallel, paranemic relationship, without helical coiling, as they undergo compaction. Bridges must then be removed during anaphase. Motivated by these findings, the present study has further investigated the process of anaphase sister separation. Morphological and functional analyses of mammalian mitoses reveal a three-stage pathway in which interaxis bridges play a prominent role. First, sister chromatid axes globally separate in parallel along their lengths, with concomitant bridge elongation, due to intersister chromatin pushing forces. Sister chromatids then peel apart progressively from a centromere to telomere region(s), step-by-step. During this stage, poleward spindle forces dramatically elongate centromere-proximal bridges, which are then removed by a topoisomerase IIα–dependent step. Finally, in telomere regions, widely separated chromatids remain invisibly linked, presumably by catenation, with final separation during anaphase B. During this stage increased separation of poles and/or chromatin compaction appear to be the driving force(s). Cohesin cleavage licenses these events, likely by allowing bridges to respond to imposed forces. We propose that bridges are not simply removed during anaphase but, in addition, play an active role in ensuring smooth and synchronous microtubule-mediated sister separation. Bridges would thereby be the topological gatekeepers of sister chromatid relationships throughout all stages of mitosis.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Human topoisomerase IIα and IIβ regulate DNA topology and knots in chromosomes during crucial cellular processes, making these enzymes common targets for anticancer drugs. However, selective inhibition of topoisomerase IIα (TOP2A) is desired to decrease adverse effects, which may be mediated by topoisomerase IIβ (TOP2B). The main region of difference between the two isoforms is the intrinsically disordered C-terminal domain (CTD), which is being studied as a target for selective inhibition. Our previous work examined several regions within the CTD to determine whether those regions impact biochemical function. In this current study, we designed and constructed four TOP2A mutants with amino acid substitutions in the CTD, which were then assessed for impact on biochemical activity. V1482D exhibited increased levels of relaxation, while both V1482D and K1520I exhibited increased levels of decatenation. No major impact on DNA cleavage or binding were observed with any of the mutants. The isolated impact of the changes on relaxation and decatenation supports the concept that the CTD can affect one aspect of the enzyme’s function in an isolated manner, which was seen in our previous study. Taken together, these results suggest that modification of specific positions within the CTD affects substrate selection. These results are mapped onto the CTD for consideration of potential regions to target for inhibition of TOP2A.

DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement. For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.

Loss of chromosome integrity is a major contributor to cancer. Checkpoints within the cell division cycle that facilitate the accuracy and outcome of chromosome segregation are thus critical pathways for preserving chromosome integrity and preventing chromosomal instability. The spindle assembly checkpoint, the decatenation checkpoint and the post-mitotic tetraploidy checkpoint ensure the appropriate establishment of the spindle apparatus, block mitotic entry upon entanglement of chromosomes or prevent further progression of post-mitotic cells that display massive spindle defects. Most of our knowledge on these mechanisms originates from studies conducted in yeast, cancer cell lines and differentiated cells. Considering that in many instances cancer derives from transformed stem and progenitor cells, our knowledge on these checkpoints in these cells just started to emerge. With this review, we provide a general overview of the current knowledge of these checkpoints in embryonic as well as in adult stem and progenitor cells with a focus on the hematopoietic system and outline common mis-regulations of their function associated with cancer and leukemia. Most cancers are aging-associated diseases. We will thus also discuss changes in the function and outcome of these checkpoints upon aging of stem and progenitor cells.

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site‐specific recombination. We show that step‐wise, site‐specific recombination by XerCD‐ dif or Cre‐ loxP can unlink bacterial chromosomes in vivo , in reactions that require KOPS‐guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD‐ dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.

PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation.

Although the Cdk inhibitor p21 Waf1/Cip1 , one of the transcriptional targets of p53, has been implicated in the maintenance of G 2 arrest after DNA damage, its function at this stage of the cell cycle is not really understood. Here, we show that the exposure of normal human fibroblasts (NHFs) to genotoxic agents provokes permanent cell cycle exit in G 2 phase, whereas mouse embryo fibroblasts and transformed human cells progress through mitosis and arrest in G 1 without intervening cytokinesis. p21 Waf1/Cip1 exerts a key role in driving this G 2 exit both by inhibiting cyclin B1–Cdk1 and cyclin A–Cdk1/2 complexes, which control G 2 /M progression, and by blocking the phosphorylation of pRb family proteins. NHFs with compromised pRb proteins could still efficiently arrest in G 2 but were unable to exit the cell cycle, resulting in cell death. Our experiments show that, when under continuous genotoxic stress, normal cells can reverse their commitment to mitotic progression due to passage through the restriction point and that mechanisms involving p21 Waf1/Cip1 and pocket proteins can induce exit in G 2 and G 1 .