topA Mutants Research Articles

Genome Instability Induced by Topoisomerase Misfunction.

Topoisomerases alter DNA topology by making transient DNA strand breaks (DSBs) in DNA. The DNA cleavage reaction mechanism includes the formation of a reversible protein/DNA complex that allows rapid resealing of the transient break. This mechanism allows changes in DNA topology with minimal risks of persistent DNA damage. Nonetheless, small molecules, alternate DNA structures, or mutations in topoisomerase proteins can impede the resealing of the transient breaks, leading to genome instability and potentially cell death. The consequences of high levels of enzyme/DNA adducts differ for type I and type II topoisomerases. Top1 action on DNA containing ribonucleotides leads to 2-5 nucleotide deletions in repeated sequences, while mutant Top1 enzymes can generate large deletions. By contrast, small molecules that target Top2, or mutant Top2 enzymes with elevated levels of cleavage lead to small de novo duplications. Both Top1 and Top2 have the potential to generate large rearrangements and translocations. Thus, genome instability due to topoisomerase mis-function is a potential pathogenic mechanism especially leading to oncogenic progression. Recent studies support the potential roles of topoisomerases in genetic changes in cancer cells, highlighting the need to understand how cells limit genome instability induced by topoisomerases. This review highlights recent studies that bear on these questions.

Abstract 3888: TOP1 mutations mediate cross resistance to ADCs in metastatic breast cancer

Abstract Background: Antibody-drug conjugates (ADCs) have improved survival for patients with metastatic breast cancer (MBC). In patients with HER2 negative MBC, two ADCs have FDA approval: sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd), both with topoisomerase-I (topo-I) inhibitor payloads. Given the many other ADCs in development, there is great interest in using ADC after ADC to maximize treatment benefit for patients. However, there is limited understanding regarding resistance mechanisms to ADCs and impact on ADC sequencing. We conducted a translational study to address this, and here we report the incidence of TOP1 mutations and clinical impact on ADC sequencing. Methods: All patients with MBC treated with ADCs at a large academic medical institution (Massachusetts General Hospital) who had comprehensive plasma-based genotyping (500 gene GuardantOMNI panel) were included. Since both SG and T-DXd have topo-I inhibitor-based payloads, we particularly focused on TOP1 mutations, variant allele frequency (VAF), and germline/somatic characterization. Incidence of TOP1 mutations in the ADC cohort was compared to The Cancer Genome Atlas (TCGA). Clinical “cross-resistance” was defined as progressive disease (PD) as best response to second ADC (ADC2) or treatment time on ADC2 of less than 60 days. Results: Based on comprehensive plasma-based genotyping we identified 4 distinct TOP1 mutations: S57C, R364H, W401C, G359E, at a frequency of 6.0% (4/67) at the time of disease progression on ADC compared to a frequency of 0.5% described in primary breast cancer in TCGA. Two of the amino acids found to be mutated are known to form direct interactions with the DNA backbone (G359) or the topoisomerase inhibitor itself (R364). For clinical resistance, one patient was briefly on ADC2 but stopped after 1 dose for toxicity; among the other 3 patients, two had cross-resistance to ADC after ADC, with both ADCs containing topo-I inhibitor payloads. Median duration on first ADC was 455 days compared to a median of 52 days for ADC2. Finally, one patient treated sequentially with 3 ADCs (all with topo-I inhibitor payloads) was found to have rising TOP1 VAF with progressive ADC treatments. Conclusion: This is the first report describing emergence of TOP1 mutations under selective pressure from ADCs and the impact on mediating cross-resistance to ADC after ADC with topo-I inhibitor payloads. Novel ADCs with alternative payloads may potentially be more effective when used sequentially after an ADC with a topo-I inhibitor. Further biomarker research is needed to optimize ADC sequencing for patients with TOP1 mutant MBC. Citation Format: Rachel Occhiogrosso Abelman, Haley Barnes, Arielle J. Medford, Annika Putur, Bogang Wu, Caroline Weipert, Geoffrey Fell, Laura M. Spring, Seth A. Wander, Beverly Moy, Andreas Varkaris, Dejan Juric, Leif Ellisen, Ryan Corcoran, Aditya Bardia. TOP1 mutations mediate cross resistance to ADCs in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3888.

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4.

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb's toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).

Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability.

Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic increase in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.

Topoisomerase I Essentiality, DnaA-Independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here, we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain (MDS42) or (ii) by an RNA polymerase (RNAP) mutation, rpoB*35, that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

Trapped topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

Rapid, direct detection of bacterial topoisomerase 1-DNA adducts by RADAR/ELISA

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

Molecular dissection of Helicobacter pylori Topoisomerase I reveals an additional active site in the carboxyl terminus of the enzyme

DNA topoisomerases play a crucial role in maintaining DNA superhelicity, thereby regulating various cellular processes. Unlike most other species, the human pathogen Helicobacter pylori has only two topoisomerases, Topoisomerase I and DNA gyrase, the physiological roles of which remain to be explored. Interestingly, there is enormous variability among the C-terminal domains (CTDs) of Topoisomerase I across bacteria. H. pylori Topoisomerase I (HpTopoI) CTD harbors four zinc finger motifs (ZFs). We show here that sequential deletion of the third and/or fourth ZFs had only a marginal effect on the HpTopoI activity, while deletion of the second, third and fourth ZFs severely reduced DNA relaxation activity. Deletion of all ZFs drastically hampered DNA binding and thus abolished DNA relaxation. Surprisingly, mutagenesis of the annotated active site tyrosine residue (Y297 F) did not abrogate the enzyme activity and HpTopoI CTD alone (spanning the four ZFs) showed DNA relaxation activity. Additionally, a covalent linkage between the DNA and HpTopoI CTD was identified. The capacity of HpTopoI CTD to complement Escherichia coli topA mutant strains further supported the in vitro observations. Collectively these results imply that not all ZFs are dispensable for HpTopoI activity and unveil the presence of additional non-canonical catalytic site(s) within the enzyme.

The Functional Consequences of Eukaryotic Topoisomerase 1 Interaction with G-Quadruplex DNA

Topoisomerase I in eukaryotic cells is an important regulator of DNA topology. Its catalytic function is to remove positive or negative superhelical tension by binding to duplex DNA, creating a reversible single-strand break, and finally religating the broken strand. Proper maintenance of DNA topological homeostasis, in turn, is critically important in the regulation of replication, transcription, DNA repair, and other processes of DNA metabolism. One of the cellular processes regulated by the DNA topology and thus by Topoisomerase I is the formation of non-canonical DNA structures. Non-canonical or non-B DNA structures, including the four-stranded G-quadruplex or G4 DNA, are potentially pathological in that they interfere with replication or transcription, forming hotspots of genome instability. In this review, we first describe the role of Topoisomerase I in reducing the formation of non-canonical nucleic acid structures in the genome. We further discuss the interesting recent discovery that Top1 and Top1 mutants bind to G4 DNA structures in vivo and in vitro and speculate on the possible consequences of these interactions.

Point mutations in topoisomerase I alter the mutation spectrum in E. coli and impact the emergence of drug resistance genotypes.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

Abstract 1742: Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases

Abstract Type II topoisomerases play key roles in transcription, replication and chromosome segregation. These enzymes modulate DNA structure by generating transient DNA breaks where the protein is covalently bound to DNA. Anti-cancer drugs targeting topoisomerase II (Top2) inhibit the religation of enzyme-induced breaks, and exert their anti-cancer effects through the generation of enzyme mediated DNA damage. In addition to small molecule inhibitors, it has been suggested that aberrant DNA structures or DNA lesions may also lead to Top2/DNA lesions. A mammalian Top2 isoform, Top2β has been suggested to play a key role in hormone-mediated transcription, and in carrying out this function, generates potentially oncogenic DNA damage. We have assessed the generation of potentially oncogenic DNA damage by Top2β in model organisms, and using the purified protein. We found that ectopic expression of Top2β is poorly tolerated in repair deficient yeast strains, while expression of the mammalian Top2α isoform lacks this property. We measured the induction of homologous recombination by Top2β in yeast and found that ectopic expression of the enzyme led to elevated levels of recombination. Recently, a de novo mutation in Top2β was reported in a patient with severe developmental delay resulting from an His63Tyr substitution in Top2β 1. We found that Top2β (His63Tyr) could not complement a defect in yeast Top2, but encoded an enzyme that was catalytically active, and induced elevated levels of recombination. Because of the inferred location of His63 in the ATPase domain of Top2β, and proximity to the ATP binding site of the enzyme, we hypothesized that the mutant protein was partially defective in ATP regulated DNA cleavage. To further explore how ATP regulates Top2β cleavage, we examined the effects of expressing a Top2β variant completely lacking the N-terminal ATPase domain of Top2β. While this mutant did not complement yeast top2 mutants, we found that it s expression induced 10-20 fold higher levels of recombination than cells expressing wild type Top2β. This result indicates that an ATP deficient variant of Top2β induces very high levels of DNA damage. We suggest that regulation of the ATPase activity of Top2β may be critical for maintaining genome stability, and may be responsible for induction of DNA damage during hormone-mediated transcription. 1. C.W. Lam et al., Global developmental delay and intellectual disability associated with a de novo TOP2B mutation. Clinica Chimica Acta 469:63-68, 2017. Citation Format: Amanda M. Johnson, Lokha Ranjani A. Boopathy, Raveena Gupta, Hannah N. Miles, Matthew Gilbertson, Karin C. Nitiss, John L. Nitiss. Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1742.

Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis.

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.

Regulatory Effect of DNA Topoisomerase I on T3SS Activity, Antibiotic Susceptibility and Quorum- Sensing-Independent Pyocyanin Synthesis in Pseudomonas aeruginosa.

Topoisomerases are required for alleviating supercoiling of DNA during transcription and replication. Recent evidence suggests that supercoiling of bacterial DNA can affect bacterial pathogenicity. To understand the potential regulatory role of a topoisomerase I (TopA) in Pseudomonas aeruginosa, we investigated a previously isolated topA mutation using genetic approaches. We here report the effects of the altered topoisomerase in P. aeruginosa on type III secretion system, antibiotic susceptibility, biofilm initiation, and pyocyanin production. We found that topA was essential in P. aeruginosa, but a transposon mutant lacking the 13 amino acid residues at the C-terminal of the TopA and a mutant, named topA-RM, in which topA was split into three fragments were viable. The reduced T3SS expression in topA-RM seemed to be directly related to TopA functionality, but not to DNA supercoiling. The drastically increased pyocyanin production in the mutant was a result of up-regulation of the pyocyanin related genes, and the regulation was mediated through the transcriptional regulator PrtN, which is known to regulate bacteriocin. The well-established regulatory pathway, quorum sensing, was unexpectedly not involved in the increased pyocyanin synthesis. Our results demonstrated the unique roles of TopA in T3SS activity, antibiotic susceptibility, initial biofilm formation, and secondary metabolite production, and revealed previously unknown regulatory pathways.

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli.

Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.

Changes of DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis mutant lacking carbon catabolite repression

Bacteria are able to prioritize preferred carbon sources from complex mixtures. This is achieved by the regulatory phenomenon of carbon catabolite repression. To allow the simultaneous utilization of multiple carbon sources and to prevent the time-consuming adaptation to each individual nutrient in biotechnological applications, mutants lacking carbon catabolite repression can be used. However, such mutants often exhibit pleiotropic growth defects. We have isolated and characterized mutations that overcome the growth defect of Bacillus subtilis ccpA mutants lacking the major regulator of catabolite repression, in particular their glutamate auxotrophy. Here we show, that distinct mutations affecting the essential DNA topoisomerase I (TopA) cause glutamate prototrophy of the ccpA mutant. These suppressing variants of the TopA enzyme exhibit increased activity resulting in enhanced relaxation of the DNA. Reduced DNA supercoiling results in enhanced expression of the gltAB operon encoding the biosynthetic glutamate synthase. This is achieved by a significant re-organization of the global transcription network accompanied by re-routing of metabolism, which results in inactivation of the glutamate dehydrogenase. Our results provide a link between DNA topology, the global transcriptional network, and glutamate metabolism and suggest that specific topA mutants may be well suited for biotechnological purposes.

Monitoring the DNA Topoisomerase II Checkpoint in Saccharomyces cerevisiae.

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control which responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.

The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis.

Topoisomerase II alleviates DNA entanglements that are generated during mitotic DNA replication, transcription, and sister chromatid separation. In contrast to mitosis, meiosis has two rounds of chromosome segregation following one round of DNA replication. In meiosis II, sister chromatids segregate from each other, similar to mitosis. Meiosis I, on the other hand, segregates homologs, which requires pairing, synapsis, and recombination. The exact role that topoisomerase II plays during meiosis is unknown. In a screen reexamining Caenorhabditis elegans legacy mutants isolated 30 years ago, we identified a novel allele of the gene encoding topoisomerase II, top-2(it7). In this study, we demonstrate that top-2(it7) males produce dead embryos, even when fertilizing wild-type oocytes. Characterization of early embryonic events indicates that fertilization is successful and sperm components are transmitted to the embryo. However, sperm chromatin is not detected in these fertilized embryos. Examination of top-2(it7) spermatogenic germ lines reveals that the sperm DNA fails to segregate properly during anaphase I of meiosis, resulting in anucleate sperm. top-2(it7) chromosome-segregation defects observed during anaphase I are not due to residual entanglements incurred during meiotic DNA replication and are not dependent on SPO-11-induced double-strand DNA breaks. Finally, we show that TOP-2 associates with chromosomes in meiotic prophase and that chromosome association is disrupted in the germ lines of top-2(it7) mutants.

Interplay between Top1 and Mms21/Nse2 mediated sumoylation in stable maintenance of long chromosomes.

Genetic information in cells is encrypted in DNA molecules forming chromosomes of varying sizes. Accurate replication and partitioning of chromosomes in the crowded cellular milieu is a complex process involving duplication, folding and movement. Longer chromosomes may be more susceptible to mis-segregation or DNA damage and there may exist specialized physiological mechanisms preventing this. Here, we present genetic evidence for such a mechanism which depends on Mms21/Nse2 mediated sumoylation and topoisomerase-1 (Top1) for maintaining stability of longer chromosomes. While mutations inactivating Top1 or the SUMO ligase activity of Mms21 (mms21sl) individually destabilized yeast artificial chromosomes (YACs) to a modest extent, the mms21sl top1 double mutant exhibited a synthetic-sick phenotype, and showed preferential destabilization of the longer chromosome relative to shorter chromosomes. In contrast, an smc6-56 top1 mutant defective in Smc6, another subunit of the Smc5/6 complex, of which Mms21 is a component, did not show such a preferential enhancement in frequency of loss of the longer YAC, indicating that this defect may be specific to the deficiency in SUMO ligase activity of Mms21 in the mms21sl top1 mutants. In addition, mms21sl top1 double mutants harboring a longer fusion derivative of natural yeast chromosomes IV and XII displayed reduced viability, consistent with enhanced chromosome instability, relative to single mutants or the double mutant having the natural (shorter) non-fused chromosomes. Our findings reveal a functional interplay between Mms21 and Top1 in maintenance of longer chromosomes, and suggest that lack of sumoylation of Mms21 targets coupled with Top1 deficiency is a crucial requirement for accurate inheritance of longer chromosomes.

Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.

Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1–PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics.

Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.

BackgroundDNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.MethodsThree SN-38 resistant (20–67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.ResultsTwo of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.ConclusionsThis study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.