Biology subjects

Berger, J. M.

Publications and source records attributed to Berger, J. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutanome-guided immunopeptidomics of blood plasma for neoepitope detection in solid tumors is constrained by cfDNA variant calling sensitivity and MS detection limits

Introduction: Neoepitopes form the basis of tumor-specific immune responses. Tissue biopsy, the primary source for neoepitope detection, is limited and invasive. Therefore, we aimed to identify neoepitopes by mutanome-guided immunopeptidomics from plasma of cancer patients. Methods: Mass spectrometry (MS) data analysis of HLA ligands from plasma (n = 4) was guided by patient-specific mutanomes of cell-free DNA (cfDNA) from plasma or tumor genomic DNA (tgDNA) from tissue. Matched tumor tissue and healthy donor plasma served as controls. Neoepitopes were validated with synthetic peptides, and immunogenicity was assessed using IFN-gamma ELISpot and intracellular cytokine staining. Results: Wild-type immunopeptidomes from tissue and plasma overlapped by 58%, with 91% of plasma HLA ligands rediscovered in tissue. 13 out of 15 tumor-associated HLA ligands detected in plasma were rediscovered in the matching tissue. However, no neoepitopes in plasma were identified by immunopeptidomics guided by cfDNA mutanomes, likely reflecting the limited overlap between cfDNA and tgDNA mutanomes (15%). Using the tgDNA mutanome as a complementary reference, two neoepitopes were detected in one patient's plasma, albeit at the MS detection limit. Both neoepitopes were also discovered in tissue, along with three tissue-exclusive neoepitopes. Two tissue-exclusive neoepitopes induced antigen-specific T cell responses in healthy donor PBMCs. Conclusion: In summary, plasma immunopeptidomics enables profiling of HLA ligands from wild-type proteins, including TAAs. In principle, neoepitope detection from plasma at the peptide level is feasible, but tissue remains the gold standard for variant calling and neoepitope identification. Improved detection methods may enable minimally invasive approaches in the future.

immunology↗

bioRxiv2026

A superfamily 1 helicase translocates on double-stranded DNA

Replicative and accessory helicases are essential for maintaining genome integrity during DNA replication and repair. Rep helicase belongs to superfamily 1 (SF1), the first helicase family solved at atomic resolution and a longstanding model for how helicases couple ATP hydrolysis to movement on DNA. Using optical tweezers combined with fluorescence imaging, we characterize how Rep translocates and navigates diverse nucleic acid and protein obstacles. We directly observe highly processive translocation on ssDNA (~300 nt/s over >13,000 nt) and show that this directional movement can drive single-stranded DNA-binding proteins (bacterial SSB, archaeal SSB, and eukaryotic RPA) along ssDNA without slowing. On encountering a short duplex barrier ([≤]60 bp), either a DNA duplex or an RNA/DNA hybrid, Rep frequently bypasses it, with termination and unwinding also observed. Unexpectedly, we identify a mode in which Rep transitions from ssDNA onto dsDNA and translocates directionally along the duplex with high processivity(>12,000bp) and at a much higher speed than on ssDNA (~1100 bp/s). AlphaFold3 modeling is consistent with Rep tracking a single strand while keeping the duplex intact. To our knowledge, this is the first demonstration of processive dsDNA translocation by an SF1 helicase, and it broadens models for how translocases operate on DNA during replication and repair.

biophysics↗

bioRxiv2026

Variant-specific nucleocapsid mutations shape host innate immune trajectories during SARS-CoV-2 infection

The SARS-CoV-2 nucleocapsid (N) protein is one of the most highly expressed proteins during infection and plays crucial roles in the protection and packaging of viral RNA, replication, suppression of the immune response and virus assembly. The N gene and the overlapping accessory open reading frame ORF9b have continually evolved throughout the circulation of the virus, likely due to ongoing adaptation to the human host. This genetic variability influences the interplay of the N protein and its interactors. Yet, little is known about how specific mutations within the N/ORF9b locus of Variants of Concern (VOCs) shape the progression and outcome of infection. Here, we use a multi-omics approach to decipher how these genetic alterations reprogram the host cell by creating Wuhan-Hu-1-based recombinant viruses carrying an isogenic backbone with respective N mutations from the VOCs (called rNs) and comparing their effects at the transcriptome and proteome levels. We found that the mutations induce distinct transcriptional and translational alterations: rN-Alpha drives a stealth-like infection characterized by sustained translation efficiency and specific evasion of the 2'-5'-oligoadenylate synthetase (OAS) innate immune sensor, whereas rN-Delta and rN-BA.2 trigger a highly elevated inflammatory response. For rN-Delta, hyperphosphorylation of the N-protein drives cellular stress culminating in necroptotic cell death. Because these viruses differ only within the N/ORF9b locus, N sequence variation emerges as a determinant of infection outcome in its own right, warranting increased surveillance attention.

molecular biology↗

bioRxiv2026

CAPRIN1 localizes to CD63-positive MVB-like SARS-CoV-2 egress compartments and limits cytopathic effects

Stress granules (SGs) are cytoplasmic condensates that assemble under cellular stress, including viral infection. The best-characterized evasion strategy in SARS-CoV-2 infection involves the viral nucleocapsid (N) protein hijacking the SG core protein G3BP1, thereby preventing SG assembly. By performing network analysis of N protein interactomes, we identified CAPRIN1 as an underexplored candidate, despite its central role alongside G3BP1 in SG assembly. Here, we provide the first high-resolution spatial and functional data on CAPRIN1 in SARS-CoV-2 infection. Using ultrastructure expansion microscopy (U-ExM), we localized CAPRIN1 and viral egress markers to enlarged, CD63- and LAMP1-positive, multivesicular body (MVB)-like egress compartments, refining the current model of SARS-CoV-2 egress. CAPRIN1 was found on both the limiting membrane of these compartments and within the intraluminal vesicles, a pattern distinct from G3BP1. Knockout of CAPRIN1 increased infection-associated cell death, promoted an aberrant cell-death phenotype, and enhanced syncytia formation. Together, our data reveal distinct activities of CAPRIN1 during infection and identify the egress compartments as MVB-like, raising the possibility that SARS-CoV-2 repurposes more than one cellular pathway for egress.

molecular biology↗

bioRxiv2026

ORC binding to Heterochromatin Protein 1 through intrinsically disordered regions is required for heterochromatin structure and function

The Origin Recognition Complex (ORC) is known for initiating DNA replication in eukaryotic cells, but increasing evidence suggests that ORC has multiple functions. Here we show that the organization and function of two major nuclear components, heterochromatin and the nucleolus, depend on multivalent interactions between Orc1 and Heterochromatin Protein 1a (HP1a) in Drosophila melanogaster. Specifically, binding requires two short motifs (R1 and R2) in the intrinsically disordered region (IDR) of Orc1, and two motifs (HGM and CTE) located in the N- and C-terminal regions of HP1a. Pairing of these four motifs promotes ORC-HP1 interactions, which also requires HP1 dimerization. Disrupting ORC-HP1a interactions by mutating the R1/R2 motifs causes defects in heterochromatin functions, specifically suppression of Position-Effect Variegation (PEV), ribosomal DNA (rDNA) decondensation, increased rRNA transcription, and nucleolar expansion, without global loss of H3K9me2 epigenetic modifications. Our findings indicate that ORC acts as a structural regulator that organizes heterochromatic regions and promotes proper genome regulation through multivalent interactions with HP1 and chromatin.

molecular biology↗

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA

Single-stranded DNA-binding proteins (SSBs) are essential for genome stability, facilitating replication, repair, and recombination by binding ssDNA, recruiting other proteins, and dynamically relocating in response to cellular demands. Using single-molecule fluorescence resonance energy transfer (smFRET) assays, we elucidated the mechanisms underlying direct strand transfer from one locale to another, protein exchange, and RNA interactions at high resolution. Both bacterial SSB and eukaryotic replication protein A (RPA) exhibited direct strand transfer to competing ssDNA, with rates strongly influenced by ssDNA length. Strand transfer proceeded through multiple failed attempts before a successful transfer, forming a ternary intermediate complex with transient interactions, supporting a direct transfer mechanism. Both proteins efficiently exchanged DNA-bound counterparts with freely diffusing molecules, while hetero-protein exchange revealed that SSB and RPA could replace each other on ssDNA in a length-dependent manner, indicating that protein exchange does not require specific protein-protein interactions. Additionally, both proteins bound RNA and underwent strand transfer to competing RNA, with RPA demonstrating faster RNA transfer kinetics. Competitive binding assays confirmed a strong preference for DNA over RNA. These findings provide critical insights into the dynamic behavior of SSB and RPA in nucleic acid interactions, advancing our understanding of their essential roles in genome stability, regulating RNA metabolism, and orchestrating nucleic acid processes.

molecular biology↗

bioRxiv2024

Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification

PCR is a cornerstone of molecular biology, but many biologically important DNA templates remain difficult to amplify. Long tandem repeats, low-complexity tracts, and sequences with extreme base composition often yield low product levels, smeared bands, stutter products, or truncated amplicons. These failures can arise because repeated cycles of thermal denaturation and reannealing promote off-register annealing, polymerase slippage, secondary-structure formation, and incomplete extension. Previously, we developed SSB-Helicase Assisted Rapid PCR (SHARP), an isothermal amplification method in which an engineered superhelicase and single-stranded DNA-binding protein replace the thermal melting step of PCR with enzymatic strand separation. Here, we tested whether SHARP can improve amplification of templates that are refractory to conventional PCR. SHARP robustly amplified up to six identical tandem repeats of the Widom 601 nucleosome-positioning sequence and up to 35 identical ankyrin repeats, targets that were poorly amplified by conventional PCR under the conditions tested. SHARP also amplified templates with extreme base composition, including a 95% AT-rich template and GC-rich templates (up to ~95% GC), as well as a 99-repeat CGG tract associated with Fragile X syndrome and a CAG/CAA repeat tract from mutant HTT exon 1 associated with Huntington disease. Together, these results show that helicase-driven isothermal amplification can expand access to repetitive and compositionally extreme DNA sequences that are challenging for conventional thermocycling-based PCR.

bioengineering↗

bioRxiv2024

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

biophysics↗

bioRxiv2024

Chromatin Buffers Torsional Stress During Transcription

Transcription through chromatin under torsion represents a fundamental problem in biology. Pol II must overcome nucleosome obstacles and, because of the DNA helical structure, must also rotate relative to the DNA, generating torsional stress. However, there is a limited understanding of how Pol II transcribes through nucleosomes while supercoiling DNA. In this work, we developed methods to visualize Pol II rotation of DNA during transcription and determine how torsion slows down the transcription rate. We found that Pol II stalls at {+/-} 9 pN{middle dot}nm torque, nearly sufficient to melt DNA. The stalling is due to extensive backtracking, and the presence of TFIIS increases the stall torque to + 13 pN{middle dot}nm, making Pol II a powerful rotary motor. This increased torsional capacity greatly enhances Pol IIs ability to transcribe through a nucleosome. Intriguingly, when Pol II encounters a nucleosome, nucleosome passage becomes more efficient on a chromatin substrate than on a single-nucleosome substrate, demonstrating that chromatin efficiently buffers torsional stress via its torsional mechanical properties. Furthermore, topoisomerase II relaxation of torsional stress significantly enhances transcription, allowing Pol II to elongate through multiple nucleosomes. Our results demonstrate that chromatin greatly reduces torsional stress on transcription, revealing a novel role of chromatin beyond the more conventional view of it being just a roadblock to transcription.

biophysics↗

bioRxiv2024

Human Topoisomerase IIα Promotes Chromatin Condensation Via a Phase Transition

Topoisomerase II (topo II) enzymes are essential enzymes known to resolve topological entanglements during DNA processing. Curiously, while yeast expresses a single topo II, humans express two topo II isozymes, topo II and topo II{beta}, which share a similar catalytic domain but differ in their intrinsically disordered C-terminal domains (CTDs). During mitosis, topo II and condensin I constitute the most abundant chromosome scaffolding proteins essential for chromosome condensation. However, how topo II enables this function is poorly understood. Here, we discovered a new and functionally distinct role for human topo II - it condenses DNA and chromatin at a low topo II concentration (100 pM or less) during a polymer-collapse phase transition. The removal of the topo II CTDs effectively abolishes its condensation ability, indicating that the condensation is mediated by the CTDs. Although topo II{beta} can also perform condensation, it is about 4-fold less effective. During the condensation, topo II-DNA condensates form along DNA, working against a DNA tension of up to 1.5 pN, greater than that previously reported for yeast condensin. In addition, this condensation does not require ATP and thus is independent of topo IIs catalytic activity. We also found that condensation and catalysis can concurrently proceed with minimal mutual interference. Our findings suggest topo II may directly participate in chromosome condensation during mitosis.

biophysics↗

bioRxiv2024

Triptolide sensitizes cancer cells to nucleoside DNA methyltransferase inhibitors through inhibition of DCTPP1-mediated cell-intrinsic resistance

While nucleoside DNA methyltransferase inhibitors (DNMTi) such as decitabine and azacitidine are effective in treating myelodysplatic syndrome (MDS)/leukemia, they have had limited utility for the majority of other cancers. Through a chemical library screen, we identified that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, or analogs significantly augmented the epigenetic and anti-cancer effects of decitabine in vitro and in vivo. These effects were attributable to inhibition of DCTPP1-mediated cleavage of 5-aza-deoxycytidine triphosphate, the convergent activated metabolite of nucleoside DNMTi, leading to enhanced drug incorporation into genomic DNA, increased DNMT degradation, enhanced global DNA demethylation and associated transcriptional reprogramming. We show that high DCTPP1 expression was associated with cell-intrinsic resistance to nucleoside DNMTi, and that triptolide and its analogs could overcome this resistance. SIGNIFICANCEWe screened a library of existing drugs to identify those capable of enhancing the anti-cancer effects of the nucleoside DNMTi decitabine. The combination of triptolide and decitabine synergistically inhibited cancer cell growth and survival in vitro, and was highly effective in inhibiting xenograft growth in vivo. Biochemical, genetic and structural biology studies with triptolide and its analogs revealed that this synergy was due to their inhibition of DCTPP1-mediated pyrophosphate cleavage from 5-aza-deoxycytidine triphosphate, the active metabolite of DNMTi. The genomic incorporation and efficacy of decitabine in cancer cell lines were significantly correlated with DCTPP1 expression more so than those of other nucleoside metabolizing genes. Triptolide and its analogs comprise rational adjuncts to nucleoside DNMTi ripe for further pre-clinical/clinical translation. HIGHLIGHTSO_LITriptolide synergistically sensitizes cancer cells to DNMTi in vitro. C_LIO_LITriptolide and decitabine combination shows favorable efficacy and safety in vivo. C_LIO_LISynergy of triptolide and decitabine is mediated through inhibition of DCTPP1. C_LIO_LIHigh DCTPP1 expression confers cell intrinsic resistance to DNMTi. C_LI

cancer biology↗

bioRxiv2023

Chromatinization Modulates Topoisomerase II Processivity

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least [~] 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a [~]100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive ([~] 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.

biophysics↗

bioRxiv2023

Using energy to go downhill - a genoprotective role for ATPase activity in DNA topoisomerase II

SO_SCPLOWUMMARYC_SCPLOWType II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II {beta} (hTOP2{beta}) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2{beta} strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavaingevitro, as well as in vivo. By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.

biochemistry↗

bioRxiv2023

Dimensional Reduction for Single Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, such studies lack the resolution needed for complex kinetic studies of multicomponent condensation. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the 2-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from 3-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a timescale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length and GC content affect threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1 and Ki-67, have much lower threshold concentration for condensation than charge-based condensing agents, with Ki-67 being the most effective as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1 and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color is used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67 mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/522433v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ed0cc9org.highwire.dtl.DTLVardef@1e26b5dorg.highwire.dtl.DTLVardef@1f6e73corg.highwire.dtl.DTLVardef@c7227b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

DNA-Stimulated Liquid-Liquid Phase Separation by Eukaryotic Topoisomerase II Modulates Catalytic Function

Type II topoisomerases modulate chromosome supercoiling, condensation, and catenation by moving one double-stranded DNA segment through a transient break in a second duplex. How DNA strands are chosen and selectively passed to yield appropriate topological outcomes - e.g., decatenation vs. catenation - is poorly understood. Here we show that at physiological enzyme concentrations, eukaryotic type IIA topoisomerases (topo IIs) readily coalesce into condensed bodies. DNA stimulates condensation and fluidizes these assemblies to impart liquid-like behavior. Condensation induces both budding yeast and human topo IIs to switch from DNA unlinking to active DNA catenation, and depends on an unstructured C-terminal region, the loss of which leads to high levels of knotting and reduced catenation. Our findings establish that local protein concentration and phase separation can regulate how topo II creates or dissolves DNA links, behaviors that can account for the varied roles of the enzyme in supporting transcription, replication, and chromosome compaction.

biochemistry↗

bioRxiv2021

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificity a priori.

biophysics↗

bioRxiv2021

Control of topoisomerase II activity and chemotherapeutic inhibition by TCA cycle metabolites

Topoisomerase II (topo II) is essential for disentangling newly replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapeutics to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies.

biochemistry↗