Note for: Roles of eukaryotic topoisomerases in transcription, replication and genomic stability

Note for: Roles of eukaryotic topoisomerases in transcription, replication and genomic stability

(doi: 10.1038/nrm.2016.111)

Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs.

Engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF–ERCC1 and MUS81).

Aim of this paper;

1.review roles of Topo in mediating chromatin dynamics, transcription, replication, DNA damage repair and genome stability.

2.dysregulation of topoisomerase -- neurodegenerative disease, immune disorder and cancer

DNA topoisomerase;

1.solve DNA topological problems

2.occur during replication and transcription

Important in both nuclear and mitochondrial genomes.

Each isoform of topoisomerase has shared and specialized roles.

Top2a and Top2b -- relax negatively supercoiled DNA

Top2a requires for chromosome segregation (dividing cell)

Top2b is indispensable for transcription in differentiated, non-dividing cells.

Activity of topoisomerase:

1.transesterification

2.reversible (preventing genome instability)

Anticancer drugs that target TOP2 enzymes induce genomic translocations that lead to secondary malignancies, and recent work suggests that TOP2β may be especially responsible for these events. Because of its danger of Topo --> therefore, the DNA damage and repair system must be standby in this regard.

Topoisomerase-induced DNA damage can have unique cellular roles and that topoisomerases may function in non-canonical ways (that is, by not only changing DNA topology), especially during transcription.

TOP1 and TOP2 enzymes do not even require DNA to be supercoiled, as they can cleave linear substrates as short as 20 bp. The two type 1B topoisomerases  have exclusive cellular localization (nuclear for TOP1 and mitochondrial for TOP1mt), whereas TOP2α, TOP2β and TOP3α are present in both nuclei and mitochondria.

Nucleosomes shield DNA from topoisomerases and restrict their reactions to linker DNA. Nucleosome-free regions, it is likely that topoisomerase activities are tightly regulated to preserve the negative supercoiling that is required for initiating transcription and replication as well as to minimize the formation of potentially deleterious cleavage complexes.

TOP1 and TOP2α are controlled by SMARCA4 (also known as BRG1), the catalytic ATPase subunit of the SWI/SNF complexes.

TOP1 is also recruited to transcriptionally active chromatin (marked by histone H3 Lys4 trimethylation (H3K4me3)) by directly binding to the facilitates chro­matin transcription (FACT) complex, which is a prom­inent histone chaperone and transcription elongation factor.

TOP2α was proposed to be a key chromosome-scaffolding protein44 and, recently, was shown to be crucial for not only for DNA decatenation but also for chromosome condensation45 and compaction of mitotic chromosomes. TOP2α sumoylation regulates its decatenating activity to ensure proper segregation of newly replicated DNA during mitosis. Through its selective recruitment to specific chromatin regions, TOP3β is emerging as a novel regulator of both transcription and translation. Chromatin modifications and chromatin-modifying complexes are likely to be key topoisomerase regulators.

Topoisomerases are required during transcription to man­age the DNA supercoiling that accumulates ahead and behind the transcription machinery. the requirements for topoisomerases differ depending on the detailed mech­anism of gene activation;

1. topoisomerase activity is required for tran­scription initiation but not for elongation

2. highly expressed genes require both Top1 and Top2, whereas Top1 alone can support genes with low transcription levels.

TOP1 tends to be kept inactive at tran­scription initiation sites to maintain negative supercoil­ing, which promotes duplex melting at promoters and transcription start sites, and that transcription pause– release requires Pol II‑mediated activation of the DNA relaxation activity of TOP1 by bromodomain-containing protein 4 (BRD4).

As these translocations are common in prostate cancer, these results suggest that TOP2β activity at promoters may have a substantial role in human tumorigenesis.

What is responsible for the high levels of breakage induced by TOP2β at gene promoters?

1. TOP2β-induced breaks could be relatively rare or accidental

2. the breaks could be long-lived. This could happen when the TOP2β re‑ligation reaction is temporarily blocked

3. TOP2β-induced breaks could be permanent. In this scenario, the enzyme has been induced by an unknown mechanism to become a nuclease (in the same way that SPO11 func­tions as a nuclease to initiate meiotic recombination)

Tyrosyl-DNA phosphodiesterase 2 (TDP2; discussed in detail below), which is a repair enzyme that removes TOP2 that is covalently bound to DNA, is required to maintain the expression of certain neuronal genes as well as genes regulated by the androgen receptor. TOP1 also forms transcription-associated, long-lived DNA breaks at androgen receptor-regulated enhancers.

The function of topoisomerases in cleaving DNA to generate a DNA damage signal and potentially per­manent DNA breaks may not be confined to promot­ers and enhancers.

TOP1 needs to be phosphoryl­ated, and a recent study showed that pause–release of Pol II requires the direct activation of TOP1 by Pol II upon the phosphorylation of Pol II by BRD4. Indeed, trap­ping TOP1 in a cleavage complex using camptothecin or its clinical derivative topotecan (see below) in cancer cells inhibits transcription elongation. Inhibition of transcription elongation by topoisomerase inhibitors has unanticipated therapeutic impli­cations.

At the transcription bubble, nascent transcripts are normally bound to their template only over a short stretch, on average 8 nucleotides or less. Longer RNA– DNA hybrids are termed R loops, and these have the capacity to stop the elongating polymerase. Poisoning of TOP1 by camptothecins also effectively leads to the forma­tion of R loops that give rise to DSBs and activate an ataxia telangiectasia mutated (ATM)-mediated DNA damage response. The mechanisms by which R loops give rise to DSBs and activated ATM are not clearly established, although they have been proposed to involve transcription-coupled nucleotide excision repair factors.

TOP1 and TOP2 enzymes have been found localized to origins of DNA replication. In mammalian cells, conditional knockdown of both TOP2α and TOP2β simultaneously did not affect rep­lication initiation or elongation. type 2 topoisomerases are not required for replication initiation. It is possible that topoisomerases need to be kept inactive at replication origins to maintain negative supercoiling and to allow priming from the single-stranded origin template.

However, it is possible that the synthetic lethality observed in cells lacking Top1 and RNase H may arise from other genotoxic properties of R loops.

As the replication fork progresses, positive supercoiling accumulates ahead of the replication fork, which can be relaxed by either TOP1 or TOP2. TOP1 acts ahead of the fork and that TOP2 acts behind the fork. Of note, TOP2α has a distinct preference for relaxation of positively supercoiled DNA63, suggest­ing that it too may be capable of acting in front of the replication fork.

Studies in budding yeast suggested that long chromosomes accumulate greater superhelical stress than short chromosomes. Of note, deletion of TOP1 results in defects in the replication of long, but not short, chromosomes. Considering that catenanes are genotoxic, these results suggest that TOP1 is also acting ahead of the fork to relax positive supercoils, thereby attenuating replication rotation and precatenane formation behind the forks.

Several studies implicate both TOP2 and TOP3 enzymes in replication termination and sister chromatid separation. Sgs1, the yeast orthologue of the human BLM helicase, promotes Top2‑mediated decatenation.RECQL5, a mammalian RecQ helicase, also facilitates human TOP2α‑dependent separation of interlocked sister chromatids by interacting with TOP2α during S phase.The interaction of BLM with TOP2α seems to be important for BLM function during homologous recombination, and it has also been suggested to be relevant for chromosomal decatenation.

In other cases — for example, when replication cannot be completed during S phase — the unreplicated chromosomal regions remain connected by UFBs, which were first identified as structures coated with BLM.

Interestingly, there seem to be several factors that participate in recruiting TOP2α to UFBs, including BLM and PICH  and probably TOPBP1. These results provide some of the strongest evidence that failure to properly decatenate chromo­somes can be oncogenic and that topoisomerases are tightly regulated by chromatin-remodelling complexes. Given the importance of topoisomerases in cell division, topoisomerase deficiency during cell division was proposed to lead to cell cycle arrest. Treating cells with the TOP2 catalytic inhibitor ICRF‑193 or its deriv­atives slows (but does not block) progression through mitosis. This observation suggested that cells might monitor the separation of catenated DNA molecules before mitosis.

After completing replication, chromosomes undergo compaction (condensation) as a prerequisite for entering mitosis. TOP2α has been identified as a main compo­nent of the chromosome scaffold. Top2 is directed to the interlinked DNA and decatenates the molecules in response to mitotic positive supercoiling, suggesting that DNA topological con­straints impel Top2 to catalyse decatenation to ensure successful cell division.

Mitochondrial DNA (mtDNA) is a circular, double-stranded genome (16.5 kb long in humans), which is packaged into DNA–protein assemblies called nucleoids that are attached to the mitochondrial inner membrane in the matrix space. Because cells contain multiple mtDNA copies (thousands in a muscle cell), mtDNA contributes up to 30–50% of the overall protein-coding genome.

The role of topoisomerases in mitochondria remains understudied and, to date, the only inhibitors of mito­chondrial topoisomerases — lamellarin D for TOP1mt and TOP1.

A lack of topoisomerase activity may result in failure to complete replication. Insufficient TOP1 activity can induce genomic breaks and could generate interference between tran­scription and replication.

The importance of topoisomerases in genomic maintenance may also explain why cancer cells, which are under replicative and transcriptional stress, frequently overexpress both nuclear and mitochondrial topoisomerases (The Cancer Genome Atlas (TCGA) data).

The selectivity of topoisomerase poisons for cancer cells remains incompletely under­stood. The main (not mutually exclusive) hypotheses are that cancer cells overexpress (TCGA data) and rely more on TOP1 and TOP2α for survival, and that DNA damage repair pathways are defective in cancer cells. Because TOP1 is covalently linked to DNA 3ʹ ends, TOP1 inhibitors act as replication poisons by generating replication-induced double-stranded ends when a replication fork collides with a trapped TOP1cc, thereby generating a ‘replication run-off ’ lesion . Replication fork reversal at these blocking sites was also proposed to generate ‘chicken foot’ lesions), which may be resolved by the MUS81–EME1 endonuclease. Another consequence of persistent TOPcc is the stalling of transcription complexes. As discussed above, TOP1 inhibitors efficiently block transcription elongation, accounting for the preferential impact of TOP1cc on long and on highly transcribed genes. Oxidative lesions have been estimated to occur at a rate of approximately 150,000 per cell per day, and base modifications, base losses and single-stranded breaks have each been estimated to occur at a rate of several thousands and up to 105 lesions per cell per day.

Two alternative pathways can remove TOPcc: the TDP excision pathway and the nuclease pathway. The removal of TOP1cc by TDP1 leaves a 3ʹ‑phosphate, which prevents TDP1 from remov­ing another nucleotide and acting as an exonuclease. However, the 3ʹ‑phosphate end cannot be readily pro­cessed by DNA polymerases and ligases unless it is first dephosphorylated by polynucleotide kinase phosphatase (PNKP). This explains the importance of PNKP and its scaffolding partner XRCC1 in the repair of TOP1cc. Among them, the most dominant pathway for TOP1cc repair in yeast and mammalian cells involves homolo­gous recombination, owing to the formation of DSBs that are produced by the collision of replication forks with TOP1cc . Indeed, TOP1cc are toxic in cells with inactivating mutations of homologous recombination components, including Rad50, Rad52 and Mre11 in yeast and BRCA1, BRCA2, XRCC2, XRCC3 and RAD52 in chicken DT40 cells. The interplay between homologous recombination, NHEJ and PARP1 remains to be clarified.

Following the removal of TOP2 from the DNA 5ʹ end and regeneration of a 5ʹ‑phosphate, direct re‑ligation of the ends can be carried out by end-joining. This directly provides a substrate for NHEJ and would be consistent with the hypersensitivity of cells deficient in the NHEJ factors KU70, KU80, ligase IV or DNA-PK catalytic subunit to TOP2cc, and with the epistatic relationship between TDP2, KU factors and NHEJ. Yet, as in the case of TOP1cc, homologous recombination also has a role in the processing of TOP2cc. the current literature regarding mammalian cells suggests a simple hypothesis: TDP1 repairs mainly TOP1cc, and TDP2 repairs mainly TOP2cc. Nonetheless, it seems likely that there may be additional specializations (perhaps at the level of recruitment) that dictate which TDP is used. The alternative pathways for the release of TOPcc rely on endonucleolytic cleavage of the DNA strand to which the topoisomerases are attached. Because these pathways excise the DNA flanking the cleavage complexes, gap-filling polymerases and homologous recombination must restore the integrity of the genome. The role of the 3ʹ‑flap endonuclease complex Rad1–Rad10 (XPF–ERCC1) in the repair of TOP1cc was characterized in yeast and human cells and reconstituted in bio­chemical systems. Similarly, the 5ʹ‑flap endonuclease XPG has been suggested to repair TOP2cc and the 3ʹ–5ʹ exonuclease Mre11 could have a role in the exci­sion of both Top1cc and Top2cc.

The choice and balance between repair by TDPs or by endonucleases require further elucidation. The ubiquitin–proteasome system appears to be required for efficient TDP activity. A potential aetiological role is suggested for TOPcc in a growing number of human pathological conditions, including cancers, neurodegenerative diseases and auto­immune syndromes.

The connection between TOPcc and cancer was first established for secondary leukae­mias induced by cancer-targeting TOP2 inhibitors. Briefly, TOP2β has been proposed as the major cause of etoposide-induced secondary leu­kaemias.

These differences (of secondary malignancy caused by TOP2cc) may originate from the cell of origin of the secondary malignancies and may reflect the differential accumulation of therapeutic small molecules in different subsets of cells. Such a mechanism could apply to other TOPcc-related cancer translocations; for example, bioflavonoids, which are present in fruits and vegetables and are TOP2 poisons, were suggested to cause childhood leukaemia. Nevertheless, the detailed biochemical processes that cause these translocations remain poorly understood. These observations provide evidence for the physiological formation of trapped TOP1cc during nor­mal development and implicate ATM, which is known to directly activate TDP1, in their removal.

The picture that has emerged is that each of these enzymes has a set of specific functions and that their specialization allows for precise coordination, especially in complicated DNA transactions that are required for replication, transcrip­tion and chromosome segregation.

Activities involve with TOPO activity (each isoform has specific role in each duty)

1.replication

2.transcription

3.chromosome segregation

within nucleus;

1.DNA repair

2.chromatin-remodelling complexes and super-enhancers

3.chromatin looping

TOP1 and TOP2ß in some contexts have different biochemical activities from what we have come to expect from a topoisomerase: instead of breakage and rapid rejoining, prolonged breakage may have biological roles.

Does the resealing of long-lasting TOP1-and TOP2β‑induced DNA breaks rely on the topoisomerase, or is the cleavage a type of DNA damage that requires other factors for repair. An exciting possibility is that other, as yet unidentified accessory proteins may regulate the cleavage–re‑ligation equilibrium of topoisomerases.

An exciting possibility is that other, as yet unidentified accessory proteins may regulate the cleavage–re‑ligation equilibrium of topoisomerases.

Dedicated strand-breaking enzymes, it is not surpris­ing that they are specifically called on when strand breaks are needed, as in the case of cutting of DNA by SPO11 for meiotic recombination.

There are two enzymes, TDP1 and TDP2, the major function of which is the release of topoisomerase–DNA adducts.

We expected that the study of the repair of topoisomerase-mediated DNA damage would teach us much about cellular responses to anticancer drugs and anticipate that this information will inform the clinical use of anti-topoisomerase agents. 

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