Topoisomerase induced DNA damage coupled diseases and therapeutic potential

Type I Topoisomerase

opoisomerase I ( op1) creates a single strand break by formation of phosphotyrosine linkage at the 5’ end of the cut end of the target DNA strand. op1 religates the break by reversing the reaction of phosphotyrosine bond formation. Relaxation of DNA supercoiling is achieved by allowing rotation of the broken strand around the op1 bound DNA strand (Stewart et al., 1998; Lesher et al., 2002; Staker et al., 2002). Religation of the DNA 5’ end requires hydroxyl group to be aligned with the tyrosine DNA phosphoester bond (Pommier et al. 2006). No A P is required for this reaction rather, the energy from breaking the phosphodiester bond is stored in the phosphotyrosine linkage and is utilized for religation (Stewart et al., 1998; Lesher et al., 2002; Staker et al., 2002). Both the type 1A and 1B are present in Mammalian system. op1A are of two types such op3α and op3β. he op1B is subdivided into op1 nuclear in nucleus and op1mt in mitochondria. op 3α is responsible for resolution of post replicative hemicatenes and recombination intermediates (Wu & Hickson, 2003; Harmon et al., 2003). op3β reduces the frequency of chromosomal translocation (Barthelmes 2000). op1 Mt couples mitochondrial functions that is translation, mtDNA expansion, A P generation and biogenesis (Dalla Rosa et al., 2014; Dalla Rosa et al. 2017). op1 nuclear is responsible for releases of supercoiling during DNA replication and transcription in nucleus (Pommier et al., 2006).

Type II topoisomerase

DNA opoisomerase II ( op2) introduces an enzyme bridged DNA double strand break (DSB) where each 5’ end of the cleaved DNA are covalently attached to the enzyme active sites by tyrosine phosphodiester linkage (Swan et al., 2022). his enzyme bridged DSB is induced by nucleophilic attack of the phosphate backbone of DNA by the op2 active site tyrosine which creates breaks forming covalent 5' phosphotyrosyl linkage between op2 and the 5’ end of the DNA (Swan et al., 2022). Enzyme then passes a second DNA duplex through the DNA break, religates the cut ends and dissociate from the DNA. opoisomerase II ( op2) requires divalent cation such as Mg2+ and A P for double strand passage reaction (Osheroff, 1986; Lindsley Osheroff et al., 1983; Lindsley Mainly implicated in DNA relaxation/decatenation and segregation (Grue et al. 1998) op2β; Mostly associated to transcription (Ju et. al., 2006) and Spo11; which expression is limited in germ cell (Swan et al., 2022). Primary structure of op2α Nitiss, 1998; Fortune Austin Berger et al., 1996). he N-terminal domain of opoisomerase II which is the A P binding site of the enzyme, homologous to the B subunit of DNA gyrase (Champoux, 2001; Velez-Cruz Berger et al., 1998). he central portion of the enzyme contains active site of tyrosine which is homologous to the A subunit of DNA gyrase (Champoux, 2001; Velez-Cruz between the two human isoforms top2α and top2β (McClendon & Osheroff, 2007). op2α plays an important role during formation of replication fork and remains tightly associated with chromosome during mitosis. (Nitiss, 1998; Bauman et al., 1997). In contrast op2β dissociates from chromosome during mitosis (Nitiss, 1998; Austin & Marsh, 1998; Woessner et al., 1991; Isaacs et al., 1998).

Top1 mediated DNA damage he key step in the catalytic cycle of op1 is the formation of a transient covalent bond between the op1 active site tyrosine and the phosphate group of DNA strand and their by a op1 DNA cleavage complex ( op1cc) intermediate is formed (Wang, 2002). Under certain conditions before relegation of the DNA strand this intermediate op1cc is stabilized that triggers a DNA damage response including DNA lesion and single strand breaks (SSBs). Stabilization of op1cc results from misalignment of the 5’ OH end of DNA. hese misalignments may be generated by any drug like camptothecin bound at the interface of the enzyme and broken DNA (Pommier & Cherfils, 2005; Pommier & Marchand, 2005). Camptothecin and op1cc binds reversibly. he ternary drug-enzyme-DNA complex and the dissociated complex establish a rapid equilibrium in pharmacological conditions. Hence, cleavage complex reverse rapidly due to dilution of camptothecin (Covey 1989). Camptothecin traps only a subset of the existing op1cc that contain a guanine at the 5’ end of the break (+1 position) (Jaxel et al., 1991; anizawa et al., 1995). Alternatively, Indenoisoquinolines stabilize those cleavage complexes which contain a cytosine at the 3’ end of the breaks (-1 position) (Antony et al., 2003).

Endogenous and frequent DNA lesions such as abasic sites, mismatches, oxidized bases, nicks at carcinogenic DNA adducts may lead to trapping of op1cc irreversibly (Pommier et al., 2006). DNA modifications due to oxidative damage can produce op1cc (Pourquier et al., 1999). he abasic site or DNA break causes irreversible misalignments of the 5’ end of the DNA and thus the op1cc induced by such lesions are irreversible (Pourquier et al., 1997; Pourquier et al. 1997a). Reactive oxygen species mediated chromatin modifications have also been reported to trap op1cc during apoptosis (Pommier et al., 2006). he irreversible op1cc referred as ‘suicide complexes’ are composed of DNA lesion where the large op1 remains covalently bound to the 3’ end of the broken DNA (Burgin et al. 1995; Shuman, 1989). he resulting disruption of the DNA backbone may lead to SSB or double strand breaks (DSBS) (Pommier et al., 2006).

Reversible op1cc may be converted into irreversible strand breaks after the DNA or RNA polymerase collide them on the leading strand during replication and on the transcribed strand during transcription respectively (Bendixen et al. , 1990; Wu & Liu, 1997). hus, both DNA & RNA synthesis convert reversible cleavage complex into permanent DNA damage. In cancer cells replication induced DNA damaged contributes to most of the cytotoxicity at low dose camptothecin while, during transcription higher dose is generally required to induce DNA damage and cytotoxicity (Holm et al. , 1989; Hsiang et al. , 1989). Camptothecin-induced op1cc may be readily converted into replication DSBs. he trapped op1cc may also inhibit transcription by blocking elongation and this is a high probability event considering that op1 is associated with transcription complexes (Pommier et al. , 1998). Single strand breaks accumulate in the DNA due to irreversible cleavage by op1 adjacent to a misincorporated ribonucleotide (Kim et al. , 2011).

Top2 mediated DNA damage op2 cuts scissile bonds on the two strands of double helix which is staggered and located across the major groove. hen enzyme form cleaved DNA molecules which contain 4 base single stranded ends at their 5’-termini (Liu & Wang, 1983; Sander & Hsieh, 1983). hus op2 covalently joins to these newly formed 5’-termini (Worland & Wang, 1989; Liu & Wang, 1983; Zechiedrich et al.,, 1989). his covalently attach enzyme-cleave DNA complex is referred to as “cleavage complex” ( op2cc) (McClendon & Osheroff, 2007). Chemotherapeutic drugs such as etoposide and doxorubicin, Xenotoxic chemicals like benzene, dietary factors (bioflavonoids) and endogenous stressor like base mismatches and apurinic sites are common op2 poisons (Loike, 1982; ewey, et al., 1984; Chen & Eastmond, 1995; Frantz et al., 1996; Strick et al., 2000;

Kingma et al. , 1997; Sabourin & Osheroff, 2000). hese types of op2 poisons stabilize the op2cc and generate DSBs formation (Long & Stringfellow, 1988). he op2 poison separates the DNA broken ends and prevents the relegation of the DNA which is cut by op2 (Pommier et al. , 2015). Many op2 poisons which are termed as interfacial inhibitors (Marchand & Pommier 2011) bind at the interface between the enzyme and DNA and then form a drug-enzyme-DNA ternary complex. Anti-tumour drugs such as doxorubicin and etoposide generate high level of op2 mediated DNA breakage. hese drugs target and trap the op2cc by stacking between the base pairs flanking the scission site and displacing the 5’phophotyrosl group from the 3’-OH group thereby preventing relegation. hus a drug-enzyme-DNA ternary complex is formed leading to DNA damage (Capranico et al. , 1990; Marchand & Pommier 2011; Pommier et al. , 1991).

op2 poisons selectively trap op2 at different sites like upstream and downstream nucleobase pairs which flank the cleavage sites where the enzymes cleave (Chen et al. , 1984; ewey et al. , 1984a; Capranico et al. , 1990a; Pommier et al. , 1991a; Capranico et al. , 1993). In mammalian cells the two isoforms of op2 are op2α & op2β. op2 poisons the anticancer drug such as Idarubicin and Etoposide target both the isoforms and stabilize op2cc preventing religation of the broken ends. Etoposide, a op2 poison, stacks between the cytosine at -1 of the break site and guanine at +5 of the break site to trap the op2βcc (Marchand & Pommier, 2011).

In op2α-DNA –drug ternary complex amino acids in the etoposide-binding pocket of op2α distinguished from op2β by Met 762 and Ser 800 in op2α while Gln 778 and Ala 816 in op2β (Nitiss & Beck, 1996). his drug stabilized op2αcc are reversible, however, their persistence leads to DSB formation (Mao et al. 2001). op2 mediated DSBs formation in the promoter region of some stimulus responsive genes in a variety of cell types and systems that are induced upon exposure to insulin, estrogen, progesterone etc. have also been reported (Ju et al. , 2006).

Human diseases caused by Top1 induced DNA damage

Top1 in tumorigenesis

Negatively supercoiled DNA facilitates RNA: DNA hybrid or R loops formation during transcription. If remains unresolved, the coiling R Loop prevents further RNA transcription & replication, leading to DSB formation (Aguilera & García-Muse, 2012). op1 interact with RNA polymerase II (RNAPII) localized at transcriptionally active region ( ARs) of the genome (Aguilera & García-Muse, 2012; Gilmour et al. , 1986). During RNAPII dependent transcription op1 supresses R loop formation by removing supercoiled DNA. he op1 holds RNAPII at the promoter proximal P site and RNAPII pauses at initiation (Khobta et al. , 2006). op1 promotes recruitment and assembly of spliceosome at ARs which phosphorylates splicing factor ( uduri et al.

2009). For coupling of RNA processing factors to ARs is critical for continuous production of full-length mature mRNA. During opoisomerase reaction with DNA, op1 covalently link with the 5’phosphate group of DNA and accidentally form op1cc which generate DNA lesion (Li & Liu, 2016). Presence of these op1cc may generate DNA damage leading to cell death or mutagenesis which is a precursor for tumorigenesis. op1 adjoining to a misincorporated ribonucleotide generally form irreversible op1cc that results into SSBs deposition (Kim et al. , 2011). In human cells novel SUMO modification at the lysine residue K391 & K436 supress the topoisomerase activity of op1 at ARs, thereby reducing op1 induced DNA damage. Any defect in the SUMOylation on K391 & K436 against op1 induced DNA damaged during transcription may turn to genome instability, mutagenesis and cancer (Li & Liu, 2016).

autoimmune antibodies against RNAPII are also frequently positive for α op1 autoantibodies (Harvey et al. , 1996). In many tissues of Scleroderma patients op1 SUMOylationis increased but op1 catalytic activity is decreased (Li & Liu, 2016). K391 & K436 residue of op1 SUMOylation supresses the activity of ranscription associated op1 while facilitate the op1-RNAPII interaction. herefore the op1 K391/K436 SUMOylation may lead to DNA damage and genome instability (Li & Liu, 2016). Hyper K391/K436 SUMOylation improve the level of op1-RNAPII complexes in cells which alter the transcriptional stress and increased programmed cell death (Li & Liu, 2016). he increased cell death due to apoptosis is expected to increase presentation of the op1-RNAPII complex to the immune system resulting into autoimmunity (Li & Liu 2016).

TOP1cc in Ataxia with Axonal Neuropathy

Accumulation of op1cc can contribute to the development of Spinocerebellar ataxia with axonal neuropathy (SCAN1) ( akashima et al. , 2002; El-Khamisy et al. , 2005; El-Khamisy et al. , 2007; Walton et al. , 2010). SCAN1 is a neurodegenerative disease ( akashima et al. , 2002). A particular mutation (H493R)

in DP1 has been identified to be the underlying cause of SCAN1 ( akashima et al. , 2002; Interthal et al. , 2001; Katyal et al. , 2007). his mutated DP1 was observed to be unable to resolve endogenous OP1ccs and as a result SCAN1 cells accumulate op1ccs. he mutation in DP1 actually inhibits the second step of DP1 mediated DNA damage repair pathway leading to accumulation of DP1-DNA catalytic intermediate or

DP1cc in addition to op1cc. he accumulation of DP1cc may block the alternative mechanisms for resolution of DNA lesions that typically respond to op1cc formation (Interthal et al. , 2005; He et al. , 2007). Endogenous accumulation of op1cc in DP1 deficient cell is prevented by treatment with transcription inhibitor (Katyal et al. 2014). herefore transcription and oxidative stress are major contributors to steady state levels of op1cc that become pathological in SCAN1.

Disease caused by Topoisomerase II induced DNA damage

Cancer therapy-related acute myeloid leukemia op2 poisons, used in cancer treatment, induces apoptosis of the cancer cells by the DNA fork collapsing and unresolved DSBs. he DSBs produced may cause mutations that promote to secondary malignancies, such as therapy-related acute myeloid leukemia (t-AML). Breast cancer and non-Hodgkin’s lymphoma (NHL) patients treated with the op2 poisons like daunorubicin etoposide, and doxorubicin, have the highest risk of being diagnosed with t-AML (Beadle et al., 2009; Morton et al., 2013; Leone, et al., 2002). he majority of t-AML cases have been diagnosed with mutation due to translocation at chromosome 11 specifically the KM 2A or MLL gene (Pedersen-Bjergaard & Philip 1991; Broeker, et al., 1996; Felix, 1998; Meyer et al., 2018). It has been observed that the MLL gene fuse with either AF9, ENL, ELL, or AF4 (Meyer et al., 2018). Some work on human hematopoietic stem cell and progenitor cells (HSPCs) demonstrated that treatment with low-dose non-cytotoxic levels of etoposide and doxorubicin increase DNA break frequency within the therapy-related breakpoint cluster region (BCR) of MLL gene ( hys et al., 2015). It has also been reported that the op2 poison etoposide may induce chromosome breakage and translocations involving MLL, AF9, AF4

AF6, and ENL in human HSPCs and lymphoblastoid cells (Gothe et al. , 2019). Altogether, the op2 and its poisons may facilitate the mutagenic process which leads to t-AML in breast cancer and NHL patients.

Papillary Thyroid Cancer (PTC)

Some evidence suggests that the OP2 also facilitates the formation of oncogenic translocations in solid tumours. According to the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database Papillary thyroid cancer (P C) increased dramatically since the 1970s (Chen et al. 2005; Davies & Welch, 2006; Enewold et al. , 2009). P C is increased by exposure of environmental chemicals and chemotherapeutic agents. Sporadic rearrangement-positive P C cases are predominantly due to chemical exposure which possess the RE /P C1 rearrangement (Nikiforov et al. , 1997; Finn et al. , 2003; Fenton et al. , 2000). he genes RE , CCDC6, and NCOA4 involved in the RE /P C1 and RE /P C3 rearrangements are located within known fragile sites indicating that they are more prone to DNA breaks than non-fragile sites of the genome (Burrow et al. , 2009). he chemical exposure-associated P C cases have been found to be triggered by the benzene and/or chemotherapeutic agents, like op1 and op2 inhibitors/poisons. Benzene is a carcinogen consisting of anti- op2 properties which is found in cigarette smoke, gasoline, and industrial emissions (National oxicology Program, 2011; Eastmond et al. , 2005). For fragile site induction, low-dose exposure from these sources is sufficient (National oxicology Program, 2011; Yunis et al. , 1987; Dillon et al. , 2012). P C has been reported in some secondary cancer patients treated with fragile siteinducing chemotherapeutic agents for cancers such as Hodgkin’s lymphoma, osteosarcoma, pediatric rhabdomyosarcoma, and others (Boffetta & Kaldor 1994, Swerdlow et al. , 1992; Goto et al. , 1996; Jimenez et al. , 1995; suchiya et al. , 1991; Verneris et al. , 2001; Yen et al. , 1993; Venkitaraman et al. , 2008; De Vathaire et al. , 1999; Gow et al. , 2003; Froelich-Ammon et al. 1994; Jonstrup et al. , 2008). H ori-3 cells when treated with low-dose, non-cytotoxic level of benzene etoposide, and doxorubicin a significant increase of the frequency of DNA breaks within the RE -BCR in intron

11 was observed (Lehman et al. , 2017). his suggests translocation process of P C is regulated by op2. Further studies shown that H ori-3 cells treated with fragile site-inducing chemicals or op1/ op2 poisons generate DNA breaks within RE intron 11 which are predominantly distributed around predicted op1 and op2 cleavage sites (Dillon et al. , 2013). As topoisomerase enzymatic activity is enhanced by DNA secondary structures, the fragility associated with RE intron 11 increase by activity of topoisomerases at the DNA secondary structures (Froelich-Ammon et al. 1994; Jonstrup et al. , 2008; Mills et al. , 2018).

Spinocerebellar Ataxia Autosomal Recessive23 (SCAR23)

he deficiency of DSB repair lead to the development of neurological abnormalities like SCAR23 (Alt & Schwer, 2018). his indicated that topoisomerase induced DNA damaged may be an important relevant lesion for neurological abnormalities. SCAR23 is an autosomal recessive syndrome that is characterized by treatment resistant epilepsy, progressive ataxia, and cerebellar degeneration (Gómez-Herreros et al., 2013; Gómez-Herreros et al., 2014). SCAR23 is also a degenerative rather than developmental disorder because SCAR23 patients also display a later age onset than other inherited ataxias, with symptoms which increases during older age (Gómez-Herreros et al. 2013; Gómez-Herreros et al., 2014). he cause of SCAR23 are mutations with DP2 and SCAR23 cells are deficient for the resolution within DP2. SCAR23 cells are insufficient for resolve the stalled op2cc and are hypersensitive to E P (epipolythiodioxopiperazine are a class of secondary metabolic toxin) (Gómez-Herreros et al., 2014). DP2 mutation results in shortened mRNA expression and nonsense mediated decay (Gómez-Herreros et al., 2014). During transcription op2cc is resolved by DP2 when DP2 insufficient in neurons show significant delay in recovery of transcription with treatment of E P (Gómez-Herreros et al., 2014). Wide expression of Genome profiling show over 100 genes which `are regulated in DP2 deficient neurons than W neurons and half of these genes are known to be epilepsy, ataxia and cognitive development (Gómez-Herreros et al., 2014). Different expressions of long genes in DP2 insufficient cells specify inhibition of topoisomerase which result in a length dependent impairment in gene expression in post mitotic stage neurons (Zylka et al., 2015).

Topoisomerase in Heterochromatin, aging and disease

Role of Top1 in Heterochromatin

Heterochromatin plays critical role in transcriptional silencing of transposons (SanMiguel et al. , 1996; Lander et al. , 2001; Waterston et al. , 2002). Aging and premature aging syndrome is caused by loss of heterochromatin (Villeponteau, 1997). Loss of heterochromatin and alteration of heterochromatin structure leads to cancer risk and neurological disease respectively (Feinberg et al. , 2016; Janssen et al. , 2018; enreiro et al. , 2014). op1 plays important role for heterochromatin structure and histone modification. Heterochromatin in Trypanosoma cruzi is unpacked by op1 inhibitors, camptothecin (CP ) and rebeccamycin (Zuma et al. , 2011). Decompression of heterochromatin with altered histone modifications is induced by the CP treatment of human HC 16 cells (Baranello et al. , 2010). Inactivation of op1 disrupts transcriptional silencing of transposons (Dinh et al. , 2015). Excess RNA-DNA hybrid or R-loop formation in heterochromatin domains was observed in op1 depleted HEK293 cells. his observation indicate important role of op1 in regulating R-loop homeostasis in heterochromatin (Manzo et al. 2018).

Topoisomerase as target for treatment of diseases

By binding opoisomerase with DNA strand create a break and passing another DNA strand through the break and releasing DNA. During cleavage process a covalent bond formed between the tyrosine residue of topoisomerase and phosphate group of breaking end of the DNA strand. opoisomerase cleaves one or both strand of the DNA double helix. he op1cc is a vulnerable intermediate that can lead to cell death if trapped by any anticancer drug or biomolecule (Seddek et al. , 2021). his property of topoisomerase may be utilizing for treatment of both infectious and noninfectious diseases (Seddek et al. , 2021).

Bacterial Top1A as target for novel antibiotics

In thermophilic bacteria op1 & op3 are found (Seddek et al. , 2021). During transcription op1 is responsible for relaxation of negative supercoiling and op3 helps in resolving replication and recombination intermediates ( erekhova et al. , 2012; erekhova et al. 2014). Endogenous inhibitors of op1 such as overexpressed n5 transposase, 4 gp55.2 and toxin YJhX have been reported to inhibit cell growth and loss of viability of bacterial cell (Seddek et al. , 2021). op1A poison inhibitors generate DNA lesion in bacteria and thereby exhibit bactericidal property (Seddek et al. 2021). he mutated bacterial op1including mutations in the conserved sequence D111, D113 and E115 triad that bind Mg²⁺ required for DNA relegation are unable to re-join the DNA (Cheng et al. , 2009). Effect of these op1 mutations is expected to mimic the action of op1A poison inhibitors. Overexpression of YjhX in E. coli leads to death even through YjhX has been shown to have no poison inhibitory effect of op1. Some bacterial pathogens such as Mycobacterium tuberculosis (Ravishankar et al. , 2015) Streptococcus pneumoniae (Liu et al. , 2017), P. aeruginosa (Yan et al. 2019) need op1 for cellular viability. Bacteria have only op1A to overcome DNA topological barrier during cellular processes and thus it is important for their survival. Catalytic inhibitors of op1 have antibacterial adequacy of the mechanism of topoisomerase inhibition. Hence bacterial op1A represents a valid target for novel antibiotics to overcome antimicrobial resistance (Wang, 2002).

of Flavivirus such as dengue and yellow fever virus (Barrows et al. , 2019). Study on role of DRD3 in viral replication have revealed that op3β is essential for replication of all single stranded DNA virus and DRD3 helps in stabilization of op3β (Prasanth et al. , 2020). herefore inhibitors specific to op3β could be used successfully as broad spectrum antiviral drugs for management of flavivirus and corona virus including SARS-COV2 infections (Prasanth et al. , 2020).

Topoisomerase as a drug target in protozoa

Camptothecin is a op1 poison which stabilizes the DNA-enzyme complex and induces slow religation (Das 2004). Camptothecin inhibits the enzyme action in kinetoplast of L. donovani , T. cruzi , T. bruci . Camptothecin also induces programmed cell death of amastigote and promastigote stage of Leishmania . Camptothecin induces oxidative stress decrease the GSH level and increased the lipid peroxidation which leads to calcium elevation from intracellular or extracellular sources (Sen et al. , 2004, 2004a). he elevated Ca²⁺ loss of membrane potential in mitochondria of the Leishmania cells. Decrease in transmembrane K⁺ level of the cell leads to apoptosis (Sen et al. , 2004, 2004a). Loss of cell membrane potential, release of cytochrome c and activation of caspase-like proteases result in apoptosis and death of parasites (Kosec et al. , 2006).

CONCLUSION opoisomerases play an important role in maintaining the topological structure of DNA in all the organisms that used DNA as hereditary material. Any malfunctioning of topoisomerase results into DNA topoisomerase covalent cleavage complex, DSB and SSB which in turn causes mutations in DNA. his mutations lead to pathogenic conditions. opoisomerase induced changes in DNA are localized in different tissues that trigger disease condition. So far the diseases reported the malfunctioning of topoisomerase has been reviewed here. However, it seems many diseases caused by topoisomerase are yet to be discovered. he topoisomerase induced DNA damaged may be a potential tool to kill harmful pathogens.

herefore attempts are being made to design drugs targeting topoisomerase of pathogens. he probability of inducing topoisomerase mediated cytotoxicity in cancers cells are also being explored by different researchers.

ACKNOWLEDGMENT:

We are thankful to Prof. Dr. imai handra Saha, Principal, Bidhannagar College forhis continuous support to review and preparation of the manuscript.. We also thank Dr. Suman Mukherjee, Assistant Professor, Department of Zoology, Bidhannagar College for his guidance, suggestion and revision of the manuscript.

CONFLICTS OF INTEREST he authors declare that they have no potential conflicts of interest.