Human topoisomerases and their roles in genome stability and organization

Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA–protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer.


Supplementary Box 1 l Topoisomerase inhibitors
Classical TOP1 inhibitors approved by the Food and Drug Administration (FDA) include two watersoluble derivatives of the plant alkaloid camptothecin (CPT): irinotecan (CPT11) and topotecan. Irinotecan is used as first-line therapy primarily for colon carcinoma, and its active metabolite is SN-38. Topotecan is used as second-line therapy for ovarian and small cell lung cancer. Non-CPT TOP1 inhibitors are being developed to avoid the severe diarrheas caused by irinotecan and drug resistance to CPT derivatives 6. In recent years, tumor-targeted delivery TOP1 inhibitors (TTTis) have been developed successfully by coupling CPT derivatives to antibodies (Antibody-drug conjugates -ADC) targeting tumor-specific antigens. The FDA-approved TTTis include Enhertu®, Trodelvy® and Onivyde® 6 . Several additional TTTis are in clinical development 6 . TOP1 inhibitors are usually given in combination with other anticancer agents. The most synergistic and rational combinations include poly(ADP ribose) polymerase (PARP) inhibitors, checkpoint kinase 1 (CHK1) inhibitors, Ataxia telangiectasia mutated (ATM) inhibitors and Rad3-like (ATR) kinase inhibitors 6 .
FDA-approved TOP2 inhibitors are among the most widely used anticancer agents [6][7][8] . They target both TOP2A and TOP2B. The podophyllotoxin derivative etoposide is widely used in the clinic. Because of its selectivity for TOP2, it is not only an anticancer drug but also a classical research tool to study how cells respond to and repair TOP2-induced DNA damage. Doxorubicin, daunorubicin and epirubicin are anthracycline derivatives discovered in fungi. In addition to TOP2 trapping activity, their potent DNA intercalating activity destabilizes nucleosomes and chromatin. They also induce oxygen radicals that damage DNA and other cellular macromolecules. Mitoxantrone is used as a secondary treatment for leukemia and for treating autoimmune diseases. The clinical use of TOP2 inhibitors is hampered by the risk of treatment-associated secondary leukemia (t-AML) (see section 3.5). Voreloxin, a quinolone derivative 10 , and CX-5461, a RNA polymerase inhibitor and DNA binder (Pidnarulex) 11 are in clinical development. In addition to the TOP2 poisons listed above, TOP2 catalytic inhibitors that act by inactivating rather than trapping TOP2 include dexrazoxane (ICRF-187), ICRF-193 and merbarone 5 .
The clinical use of TOP1 and TOP2 remains empirical. High expression of TOP1 and TOP2 has been proposed to account for the response but is not implemented clinically. Recently, the expression of Schlafen 11 (SLFN11) has been identified as a predictive genomic biomarker for both TOP1 and TOP2 inhibitors, as well as PARP inhibitors and platinum derivatives 7 . Clinical validation of SLFN11 as a predictive biomarker is ongoing [8][9][10] .
In addition to the therapeutic value of targeting human topoisomerases for cancer therapy, targeting the bacterial type II topoisomerases with fluoroquinolone derivative (ciprofloxacin, levofloxacin) is among the most effective therapies for a broad range of infectious diseases 11 .
Topo-Seq 14 , like ChIP-Seq uses topoisomerase antibodies to pull-down the topoisomerase but skips the crosslinking step to retrieve exclusively the covalently bound topoisomerase complexes engaged in the TOPccs.
End-Seq directly maps the break sites at nucleotide resolution 22 . It includes an end-processing step with ExoVII nuclease to cleanse the 5'-ends of the breaks following their proteolytic digestion 22,23 .
Both End-Seq and Topo-Seq detect TOPccs but exclude topoisomerase sites where topoisomerases are associated with chromatin non-covalently.
In addition, a novel NGS method (SAR-seq) has revealed that both TOP2cc and TOP1cc generate unscheduled DNA synthesis within expressed genes, providing insights into the DNA repair step induced by TOP1/2cc 24 .
A variation of the End-Seq method skipping the proteolytic step during sample preparation can detect the TOP2ccs that have been processed to frank breaks (protein-free DSBs) 25 .
Etoposide is generally used to map genomic TOP2 sites 22,[25][26][27] . However, etoposide only traps a subset of the TOP2ccs 8,28 (Supplementary Box 1) due to their interfacial inhibition mechanism, which involves specific bases pair sequences flanking the cleavage sites 8,9 . Moreover, TOP2 inhibitors trap both TOP2A and TOP2B relatively indescrimately 6,8 . Studies with self-poisoning topoisomerases may provide complementary approaches to map TOP2ccs 29 , TOP3Bccs 30 and TOP1MTccs 31 . To our knowledge, End-Seq techniques have not been developed to map TOP1ccs and TOP3Bccs at nucleotide resolution.
Supplementary Fig. 1 l Biochemistry of human topoisomerases. a l Reversible DNA cleavage by type IB topoisomerases (TOP1 and TOP1MT). Two excision enzymes hydrolyze the tyrosyl-DNA linkage, TDP1 and APE2, which leaves 3'-phosphate and -hydroxyl ends, respectively. b l Reversible cleavage by type II and type III topoisomerases (TOP2A, TOP2B, TOP3A, TOP3B)  Data were retrieved from the CCLE (Cancer Cell Line Encyclopedia) database of 1,019 difference cancer cell lines using CellMinerCDB (https://discover.nci.nih.gov/cellminercdb) 33 . Snapshots of selected gene correlation with TOP2A are shown.