Top II enzymes are essential in the elongation of replicating DNA chains and during segregation of newly replicated chromosomes. During semiconservative replication, the replisomal machinery at the advancing replication fork forces the intertwined DNA strands ahead of it to become overwound or positively supercoiled. These positive supercoils are redistributed behind the fork by the rotation of the replicative machinery around the helical axis of the parental duplex.
This causes intertwinement of the newly replicated DNA double helices, forming precatenanes [ ]. As replication nears completion, the unreplicated double-stranded DNA dsDNA segment in between the converging forks becomes too short for the action of a type I topoisomerase and replication must first be completed resulting in intertwinement of the replicated duplexes into catenated dimers [ , ].
These intertwines are decatenated by the action of Top II to allow for chromosome separation in mitosis [ , ]. In yeast, which only possesses a single type II topoisomerase, loss of the enzyme results in chromosome nondisjunction, accumulation of catenated dimers, and inviability at mitosis [ , , ]. In contrast, it has been shown in human cells that Top IIa but not Top IIb is essential for chromosome segregation [ ]. Transcription of nascent mRNA presents topological obstacles akin to those seen during replication [ ].
Transcription elongation is accompanied by changes in the local supercoiled state of DNA, forming positive supercoils ahead and negative supercoils behind the transcriptional machinery [ , , , ]. While loss of Top I or Top II alone in yeast generally does not compromise transcription, top1 top2 temperature-sensitive double mutants exhibit significant inhibition of rRNA synthesis and, to a lesser extent, mRNA synthesis at nonpermissive temperatures [ ].
In addition, double mutants accumulate negative supercoils in extrachromosomal plasmids which appear to promote transcription initiation of ribosomal minigenes on such plasmids [ , ].
More recent studies of yeast rRNA synthesis suggest distinct roles of Top II and Top I, acting, respectively, to relieve positive supercoiling ahead of and negative supercoiling behind the advancing polymerase [ 11 ]. Transcription has long been known to stimulate HR in a locus-specific manner [ — ] and it is emerging that physiological DSBs may be involved in regulated transcription [ , ].
Furthermore, it has recently been found that DSBs induced at transcriptionally active sites in human cells preferentially promote HR over NHEJ and this repair is dependent on the transcription elongation-associated epigenetic marker H3K36me3 [ ]. It is thus becoming increasingly clear that transcription and HR-dependent DSB repair may be reciprocally regulated. Top IIb-mediated DSB formation in a target promoter has also been shown to be required for glucocorticoid receptor-dependent transactivation [ ].
More recently, the spotlight has shifted to the broadening role of activity-induced DSBs in neuronal physiology and neuropathology. This increase was not observed with other DSB-inducing agents including neocarzinostatin, bleomycin, and olaparib, suggesting a Top II-mediated mechanism [ 12 ]. Together these indicate a role of DSBs acting within target gene promoters in the regulation of nIEG transcription [ 12 ].
This corroborates recent evidence supporting DSB induction in mouse neurons in response to environmental stimuli in the form of spatial exploration [ ]. These DSBs were found to predominate in the dentate gyrus suggesting a physiological role in spatial learning and memory [ ]. Overall, these results suggest that neuronal activity specifically induces targeted DSBs involved in transcriptional regulation of nIEGs.
Interestingly, studies of repair kinetics of DSBs generated in the Fos promoter following NMDA treatment demonstrated long-lived breaks, being repaired within 2 hours of stimulation. However, the significance of this extended lifespan is as yet unclear [ 12 ]. CTCF organises genomic superstructure, defining chromatin architecture to control long-range interactions between different genomic loci.
Major effects of CTCF action include the establishment of chromatin domains and blockade of promoter-enhancer interactions [ ]. Combining these results Madabhushi et al. It was recently postulated that activity-induced DSBs in nIEGs lead to genomic rearrangements involving transposable elements producing a distinct form of somatic mosaicism in neurons, with implications on plasticity, network formation, and potential interplay with age-related epigenetic changes and disease [ ].
Additional mechanistic complexity derives from the need for concerted repair of activity-induced DSBs. There is growing evidence suggesting that aberrant Top II activity may play a role in the aetiology of neurodevelopmental defects. Recent work involving clinical whole-exome sequencing identified a novel TOP2B mutation associated with intellectual disability, autistic traits, microcephaly, and developmental retardation [ ] and homozygous mutations in TDP2 have been linked with cases of intellectual disability, epilepsy, and ataxia [ 19 ].
Top IIb has been shown to be important in neuronal differentiation and survival and is upregulated during the transition from mouse embryonic stem cells to postmitotic neurons with reciprocal downregulation of Top IIa. Furthermore, loss of Top IIb in mice results in premature degeneration and apoptosis of neurons at later stages of differentiation concomitant with transcriptional deregulation of genes involved in neurogenesis and cell division [ 13 , 14 ].
Pharmacological inhibition of Top I in mouse cortical neurons results in preferential downregulation of long genes, many of which are associated with autism spectrum disorder.
Furthermore, it has been proposed that environmental and dietary sources of topoisomerase poisons and inhibitors in early neurodevelopment could be a potential cause of autism and there have been calls to factor this into food safety and risk assessments [ , ].
Together, these findings point towards the importance of DSB repair regulation in neuronal physiology and activity-induced DSB handling, but further studies will be required to elucidate more specific roles of activity-induced DSBs in neuropathology. Studies of isolated chromosomes reveal association of one Top II molecule per 23, base pairs of DNA, largely at and around loop bases, suggesting a structural role in higher-order DNA architecture [ , ].
Indeed, Top II binding and DSB formation sites have been identified in matrix-attachment regions, cis- elements which are involved in long-range chromatin organisation [ , ]. However, photobleaching experiments have revealed both Top II isoforms to be highly mobile in the chromosomal scaffold, arguing for a more complex dynamic interaction with chromatin structure [ ].
DNA topology and chromatin structural changes are known to be intricately related [ ]. Early work with temperature-sensitive top2 yeast [ ] and complementation experiments with Top II-immunodepleted Xenopus egg extracts [ ] revealed Top II to be important in chromosome condensation and segregation in mitosis, presaging the discovery of a more active role of the enzyme in chromatin remodelling.
Furthermore, Top II-mediated DNA supercoil relaxation is stimulated by the Drosophila protein Barren [ ], which is involved in chromosome condensation and disjunction [ ]. ChIP experiments in S. Thus, it appears that Top II DSB handling is important in highly diverse manipulations of chromatin packing and further studies are needed to elucidate the precise regulation thereof.
As in genomic DNA, replication and transcription processes exert topological stresses on mtDNA molecules which are relieved by native topoisomerases [ , ]. Although mitochondrial Top IIb is C-terminally truncated compared to its nuclear counterpart, it still retains basic type II topoisomerase DSB turnover activity, albeit with reduced processivity, and exhibits a similar pharmacological profile [ ]. Top1mt deletion in mice results in elevated mtDNA transcription and induction of a stress response accompanied by considerable upregulation of Top IIb, suggesting a potential compensatory role of the type II enzyme [ ].
This picture is further complicated by the recent discovery of active full-length Top IIa and Top IIb in both murine and human mitochondria, which, although as yet uncharacterised, are likely involved in classical functions in replication and transcription like their nuclear counterparts [ ].
Top IIIa is a type IA topoisomerase which is found in both the nucleus and mitochondria and has been shown to play a crucial role in the maintenance of mtDNA genomic integrity in Drosophila. Indeed, loss of mitochondrial import of Top IIIa in Drosophila is associated with a reduction in mtDNA copy number, mitochondrial dysfunction, impaired fertility, and accelerated ageing [ , ].
A recently proposed but unexplored theory suggests that mitochondrial Top II may be involved in ageing [ ]. These deletion events are found to contribute to dysfunction of the respiratory chain [ ] and other tissue impairments associated with ageing [ — ].
Mammalian mitochondrial Top IIb has been found to be inhibited by the anticancer drugs amsacrine and teniposide [ ] and it is possible that various known nuclear Top II poisons, ranging from the fungal toxin alternariol [ ] and chemotherapeutics [ ] to dietary components such as bioflavonoids from fruit and vegetables [ ], may also inhibit the truncated or full-length type II topoisomerases in mitochondria.
Furthermore, base oxidations, abasic sites, and other exogenously induced base modifications can act, via an unknown mechanism, to abnormally enhance Top II DSB formation [ ] and promote accumulation of DNA-enzyme intermediates at sites of damage [ ]. While it is likely that mitochondrial Top II performs similar DSB-related functions to its nuclear counterpart, further studies will be needed to elucidate the precise nature of its role in mitochondria and its contribution if any to the ageing process.
Further insight into the role of Top II-induced DSBs in mitochondria may be drawn from parallels in Kinetoplastids, a group of flagellated protozoa which includes the parasitic Trypanosoma and Leishmania species, which possess a characteristic dense granule of DNA called a kinetoplast within their single mitochondrion.
The kinetoplast consists of an enormous network of catenated circular mtDNA kDNA comprising short minicircles and much larger maxicircles and thus represents a considerable topological challenge in replication and segregation [ ]. The mitochondrion contains a type II topoisomerase which localises to the kDNA [ , ] and has been shown to be important for the maintenance of structural integrity in the kinetoplast [ ], mending of holes in the kDNA following individual minicircle release for replication [ ], segregation of daughter minicircles [ ], and their reattachment to the kDNA network [ ].
DSB physiology is thus, at its very core, a molecular surgery, reliant on both calculated incisions and timely suturing thereof, all the while, under the intense scrutiny of numerous regulatory mechanisms. While the basic biochemical mechanisms of the processes discussed above are well established, their multilateral spatiotemporal regulation remains a field of active research.
The roles of recombination defects in human disease and tumorigenesis are being increasingly appreciated and further studies will be needed to examine the nature of the dysregulation of DSB physiology in pathogenesis. Novel tissue-specific roles of topoisomerases in neurophysiology are only just being uncovered with future studies required to elucidate the function of DSBs therein to understand mechanisms of learning, memory, and cognition. In addition, future work is necessary to characterise the nature of mitochondrial DSB physiology and the as yet unexplored implications on mitochondrial metabolism, disease, and ageing.
Furthermore, both topoisomerases and cancer DSB repair deficiencies are important areas of investigation in the development of anticancer therapeutics, and an understanding of the physiological roles of these targets in multiple disparate processes will inform the design of specific inhibitors with fewer off-target effects.
This account illustrates the breadth of DSB physiology across cellular biology and adds a new dimension of complexity to the regulation of genomic transactions on a basic biochemical level. The emerging role of DSB-dependent changes in topology, not only in replication and basal transcription, but also in activity-induced transcriptional changes, and potential roles in the chromatin landscape and mitochondria paint a picture of a far more active role of DSBs in fundamental DNA metabolism than previously thought.
The authors are very grateful to Dr. Joseph Maman for his guidance and support during the production of this work and for his ever helpful advice whenever needed. Khan and Syed O. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Journal overview. Special Issues. Khan 1 and Syed O. Academic Editor: Shigenori Iwai. Received 08 Jul Accepted 24 Sep Published 18 Oct Abstract Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Introduction Just as DNA breakage can devastate genomic integrity, it can also be deliberately and precisely exploited by cells in feats of genetic craftsmanship.
Figure 1. Summary of the diverse roles of physiological DSBs in biological processes. References M. Jackson and J. Schipler and G. Jeggo and M. Brandsma and D. Xu, H. Zan, E. Pone, T. Mai, and P. Lam and S. Lucas, T. Germe, M. Chevrier-Miller, and O. Baxter and J. French, M. Sikes, R. Hontz et al. Madabhushi, F. Gao, and A. Tiwari, L. Burger, V. Nikoletopoulou et al. E—E, Lyu, C. Lin, A. Azarova, L. Cai, J. Wang, and L. Thakurela, A.
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Now, in eLife , Susan Rosenberg of Baylor College of Medicine and colleagues—including Chandan Shee as first author—have provided a promising new tool for the study of one such insult: the double strand break Shee et al. Double strand breaks are considered the most dangerous of all the DNA lesions.
If left unrepaired, the resulting chromosome discontinuity often results in death. There are two main ways to repair a double strand break. Recombinational DNA repair is accurate but it relies on the presence of an unbroken homologous chromosome. Non-homologous DNA end-joining, on the other hand, repairs the break, but usually at the expense of adding or deleting genetic information Chapman et al. Dangerous as they are, double strand breaks are sometimes deliberately introduced into a chromosome.
In yeast, directed double strand breaks are a prelude to an intrachromosomal exchange of genetic information that produces a mating type switch Haber, Double strand breaks are also central to genetic elements called transposons, and in genomic rearrangements that are integral to the immune system. Accurate real-time detection of double strand breaks in a cellular genome is thus of great interest in the continuing effort to understand genome maintenance and function.
A variety of techniques have been developed to detect and quantify double strand breaks, but they all have one or more deficits in terms of utility, efficiency, sensitivity or specificity.
Shee, Rosenberg and colleagues—including co-workers from the University of Texas, the MD Anderson Cancer Center and the University of Minnesota—now report a new approach, based on a protein called Gam, that offers some substantial advantages over existing approaches Shee et al.
Gam is encoded by the bacteriophage Mu: this is basically a hybrid of a bacterial virus and a transposon, and it makes a living by moving efficiently within and between bacterial genomes Baker, ; di Fagagna et al. When an integrated genomic copy of Mu replicates and transposes, the Gam protein protects the free ends of the Mu chromosome as they are transiently exposed.
Gam is related to two eukaryotic proteins, Ku70 and Ku80, that are involved in non-homologous DNA end-joining. Whereas the Ku proteins bind to double strand ends, they also interact with an array of other eukaryotic proteins and DNA structures, rendering them less useful for development of a general reagent that binds to double strand breaks.
Gam is a simpler system, a single protein with a high affinity for double stranded ends. When this protein is expressed in a cell, the double strand breaks light up when the cell is illuminated, and this allows the number of breaks to be counted.
For example, the double strand breaks that occur during DNA replication can be pinpointed Figure 1 , as can the sites where the restriction enzyme Scel cleaves a particular chromosome.
Shee et al. Many protocols are being developed, so with some creativity and luck, it is possible to design a variation of the technique to suit a particular experiment. This enzyme is a template-independent polymerase that can add nucleotides to free 3' DNA ends with free 3'OH groups. If there are clean DNA breaks, these damaged chromosomal ends will be labeled with fluorescent nucleotides. The treated samples are resolved by PFGE, and the gel is scanned to detect fluorescence.
The fluorescent smear observed in the treated samples results from specific labeling of DNA breaks with fluorescence. One advantage of this method is that there is no background due to intact chromosomal DNA Figure 3B; compare lanes 3 and 4 with control lane 1.
With this technique, the difference between the treated and untreated samples becomes more evident compare Figure 3A with Figure 3B. There are still many discrepancies in the field of DSB repair regarding which factors are involved in the processing of DNA ends.
The types of breaks being generated to study DSB repair differ depending on the agent used. IR or reactive oxygen species induce what is referred to as DSBs with "ragged" ends, which means that the ends need to be trimmed down before they can be repaired by a polymerase or be ligated.
Another limitation of inducing DSBs with a radiomimetic drug, IR, or other chemical agent is that the damage occurs randomly.
If we want to study the detailed events taking place at each break, we need a different experimental system. For example, in yeast, an inducible endonuclease system, was developed. In this system, a single DSB can be induced synchronously in a large number of cells, allowing the mechanism of DSB repair to be studied in more detail at the molecular level. Despite some answers about mechanism, a slew of unanswered questions remains.
For instance, how does the degradation of DNA ends take place? Does the extent of degradation have an effect on the repair pathway chosen? How is the degradation of DNA ends regulated?
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