Packaging of genomic DNA by proteins and super coiling into chromatin and chromatin-like structures (in bacteria) influences nearly all nuclear process such as replication, transcription, repair, and recombination. A ubiquitous class of enzymes termed “DNA topoisomerases” pay key roles during these process. The reactions catalyzed by the members of the DNA topoisomerases family share a common chemistry, which involves phosphodiester bond breakage and re-joining, to bring about a change in the linking number of DNA. Nevertheless, the underlying mechanisms used by these enzymes differ significantly from another. Consequently, DNA topoisomerases are divided into type I and type II enzymes. The mechanism(s) by which DNA topoisomerases perform their functions, and act as targets for anti-bacterial and anti-neoplastic drugs, has attracted considerable interest. Based on these and other finding, I have chosen DNA gyrase from mycobacteria as the subject of my Ph.D. theses investigation. The prokaryotic enzyme, DNA gyrase, is unique amongst all topoisomerases being the only enzyme capable of introducing negative super coils in to duplex DNA. Since no equivalent enzymatic activity has been reported in humans, this essential enzyme has been exploited as a during target against many microbial infections including tuberculosis.DNA gyrase is a tetrameric protein, comprised of two pairs of subunits, encoded by gyrA and gyrB. Inhibitors of DNA gyrase know till date target either of the two subunits and are categorized broadly in to two class, viz. coumarins and quinolones. With the emergence of multiple-drug resistant strains of pathogenic bacteria such as Mycobacterium tuberculosis, which is a leading cause of death world-wide, there is a need to develops new lead molecules with novel mechanisms of inhibition. Towards this end, a new approach to inhibit the mycobacterial DNA gyrase using single-chain antibody has been explore in the present study. In addition to this, the differences in the catalytic properties of the subunits and assembly of the Mycobacterium smegmatis enzyme vis-à-vis Escherichia coli DNA gyrase have been examined. Further, the in vivo relationship of DNA gyrase with the transcription machinery of the cell has also been investigated, with an emphasis on the biology of mycobacteria.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

A steady state level of negative supercoiling is essential for chromosome condensation, initiation of replication and subsequent elongation step. DNA gyrase, found in every eubacteria, serves the essential housekeeping function of maintenance of the negative supercoiling status of the genome. The functional holoenzyme is a heterotetramer, comprising of two GyrA and two GyrB subunits. DNA gyrase is an indispensable enzyme and serves as a readily susceptible target for natural antibacterial agents. The enzymatic steps of topoisomerisation by gyrase involve transient double strand break and rejoining of the strands after intact duplex transfer. Corruption of its catalytic cycle can lead to the generation of cytotoxic double-strand DNA breaks. Most of the anti-gyrase agents achieve their objective by targeting the vulnerable step of the reaction cycle i.e. DNA cleavage step. Bacteria on their part must have evolved and adopted strategies to counter the action of external agents and prevent the generation of double strand breaks thereby safeguarding their genome. In the present thesis, attempts have been made to understand the role of three endogenous gyrase interacting proteins in gyrase modulation and cellular defense against anti-gyrase agents. The thesis is divided into six chapters. Chapter 1 introduces the wonder enzymes “DNA topoisomerases” starting with a brief classification of these enzymes and their physiological functions. In the next section, DNA gyrase has been discussed in greater detail. The structural aspects as well as the mechanism of the topoisomerisation reaction catalyzed by gyrase have been discussed. Final section gives an overview of different gyrase modulators known till date focusing on their source, structure and mode of action. The scope and objectives of the present study is presented at the end of this chapter. In Chapter 2 is aimed at understanding the physiological role of GyrI. GyrI, originally identified in Escherichia coli as an inhibitor of DNA gyrase, has been previously shown in the laboratory to render protection against gyrase poisons and also various other DNA damaging agents (mitomycin C, MNNG). Abolishing GyrI expression renders the cell hypersensitive to these cytotoxic agents. Interestingly, GyrI exhibits contrasting behavior towards two plasmid encoded proteinaceous poisons of DNA gyrase. It reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin. However, a positive cooperation between GyrI and F plasmid encoded toxin CcdB, results in enhanced DNA damage and cell death. These results suggest a more complex functional interplay and physiological role for GyrI. Search for other chromosomally encoded gyrase inhibitors led to YacG, a small zinc finger protein (7.3kDa) from E. coli, shown to be a member of DNA gyrase interactome, in a protein-protein interaction network described recently. Chapter 3 deals with the detailed characterization of YacG. It is shown that YacG inhibits DNA gyrase by binding to GyrB subunit and preventing DNA binding activity of the enzyme. More importantly, it protects against the cytotoxic effects of other gyrase inhibitors like ciprofloxacin, novobiocin, microcin B17 and CcdB. Further investigations revealed that YacG and its homologues are found only in proteobacteria. Hence, it appears to be a defense strategy developed by gram-negative bacteria to fight against the gyrase targeting cytotoxic agents. Inhibition by YacG appears to be specific to E. coli gyrase as mycobacterial enzyme is refractile to YacG action. GyrB, only in gram-negative organisms, possesses extra stretch of 165 amino acids, indispensable for DNA binding. Biochemical experiments with the truncated GyrB lacking the extra stretch reveal the importance of this stretch for stable YacG-GyrB interaction. E. coli topoisomerase IV is also resistant to YacG mediated inhibition, probably due to the absence of the extra stretch in ParE subunit, which is otherwise highly similar to GyrB. Further, YacG homologues from other proteobacterial members (Sinorhizobium meliloti and Haemophilus influenzae homologues sharing 35% and 63 % identity with E. coli YacG respectively ) also inhibits E. coli DNA gyrase at comparable levels. YacG thus emerges as a proteobacteria specific inhibitor of DNA gyrase. The occurrence of both YacG and the gyrase extra stretch only in proteobacteria, suggest co-evolution of interacting partners in proteobacteria. In Chapter 4, the study of endogenous gyrase modulators is extended to Mycobacterium sp. glutamate racemase (MurI) from E. coli has been shown earlier to be an inhibitor of DNA gyrase. However, nothing much was known about its mode of action. MurI is an important enzyme in the cell wall biosynthesis pathway, which catalyses the conversion of L-glutamate to D-glutamate, an integral component of the bacterial cell wall. In this chapter, it is demonstrated that M. tuberculosis MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species specific as E. coli gyrase is also inhibited. However, it is gyrase specific as topoisomerase I activity remains unaltered. The mechanism of inhibition by MurI has been elucidated for the first time and it is shown that MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. Chapter 5 is the extension of the studies on glutamate racemase into another species, i.e. Mycobacterium smegmatis. DNA gyrase inhibition seems to be an additional attribute of some of the glutamate racemases, but not all, as Glr isozyme from B. subtilis has no effect on gyrase activity in spite of sharing a high degree of similarity with the gyrase inhibitory glutamate racemases. It is shown that like the M. tuberculosis MurI, M. smegmatis enzyme is also a bifunctional enzyme. It inhibits DNA gyrase in addition to its racemization activity. Further, overexpression of the enzyme in M. smegmatis provides protection to the organism against fluoroquinolones. DNA gyrase inhibitory property thus appears to be a typical characteristic of these MurI and seems to have evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double strand breaks in the genome. In the above chapters, it is shown that besides its crucial role in cell wall biosynthesis, mycobacterial MurI moon lights as DNA gyrase inhibitor. That the two activities exhibited by M. tuberculosis MurI are unlinked and independent of each other is demonstrated in Chapter 6. Racemization function of MurI is not essential for its gyrase inhibitory property as mutants compromised in racemization activity retain gyrase inhibition property. MurI- DNA gyrase interaction influences gyrase activity but has no effect on racemization activity of MurI. MurI expression in mycobacterial cells provides protection against the action of ciprofloxacin, thereby suggesting a role of MurI in countering external agents targeting DNA gyrase. Further M. tuberculosis MurI overexpressed in near homologous expression system of M. smegmatis yields highly soluble enzyme which can be further used for structural and functional studies. In conclusion, the studies reveal that the endogenous inhibitors essentially influence the enzyme activity by sequestering the enzyme away from DNA. None of them cause cytotoxicity, which usually arises as a result of DNA damage caused by accumulation of gyrase-DNA covalent intermediate. On the contrary they provide protection against such gyrase poisons. Comparative analysis of these proteinaceous inhibitors, however, does not reveal a common motif or structural fold, required for their ability to inhibit DNA gyrase. Based on these studies, it can be proposed that these endogenous proteins exist to serve as cellular defense strategies against external abuse and also to modulate the intracellular activity of DNA gyrase as and when required, for accurate division, functioning and survival of the cells.

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.