Biology subjects

Nudler, E.

Publications and source records attributed to Nudler, E..

18 recordsLinked to original sources

bioRxiv2026

H-NS silences antiviral immunity through 3D chromatin compaction

Bacterial antiviral immunity systems can be toxic to their hosts and must be tightly regulated. Immunity genes are often encoded by AT-rich mobile genetic elements subjected to silencing by nucleoid binding protein H-NS. Using RNA-seq, ChIP-seq, and chromosome 3D reconstruction with Micro-C, we find that H-NS binds and compacts bacterial immunity loci. H-NS deletion reveals anti-phage activity in model Escherichia coli strains generally considered phage-sensitive. Extending this approach to environmental isolates, we show that removal of H-NS silencing enhances defense, allowing bacteria to restrict phages with anti-defense proteins. We also discover Madara, a novel immunity system that complements the co-regulated BREX defense. Our results establish H-NS as a master regulator of bacterial immunity and highlight the importance of expression levels for anti-phage activity in native hosts.

molecular biology↗

bioRxiv2026

OLD sentinel: an abortive tRNase surveys phage replication and DNA defects in RecBCD-compromised cells

OLD, an abortive immunity protein from prophage P2, consists of an ABC ATPase sensor and a TOPRIM nuclease effector -- a core architecture shared by a large protein family, including components of antiphage systems Gabija, PARIS, Septu, and Lamassu. OLD was originally identified for its lethality in recB-deficient cells and inhibition of bacteriophage {lambda} infection, but the mechanisms governing its activation have remained elusive. Here, we present the cryo-EM structure of an inactive OLD tetramer and show that destabilization into dimeric form opens the TOPRIM catalytic site, stimulating tRNA cleavage. This activity arrests translation, a phenotype rescued by phage-encoded tRNAs. We demonstrate that OLD activation is not strictly RecBCD-dependent: OLD binds aberrant DNA structures in recB-deficient cells, but activation during infection requires recognition of single-stranded DNA hairpins at the phage replication origin. Collectively, our findings reveal how host and phage DNA processing factors create a complex landscape controlling OLD-mediated immunity.

molecular biology↗

bioRxiv2026

ZCWPW1 organizes telomeric architecture to drive meiotic chromosome movements

Meiotic homolog pairing relies on programmed DNA recombination and large-scale chromosome movements, yet, how these genetic and mechanical events are coordinated remains unclear. ZCWPW1 is a histone reader that recognizes PRDM9-deposited chromatin marks. We identify an unexpected role for ZCWPW1 as a regulator of rapid prophase movements (RPMs). Using super-resolution imaging, we show that ZCWPW1 is strongly enriched at subtelomeric regions of mouse spermatocytes, where it stabilizes TRF1, LINC complex components, dynein, and meiosis-specific cohesin (STAG3). Loss of ZCWPW1 disrupts telomere architecture, weakens telomere-LINC- motor coupling, and abolishes chromosome movement, leading to defective synapsis and pairing, and persistence of DSBs. These defects are more severe than, and mechanistically independent of, those observed in Prdm9-/- spermatocytes. Together, our findings reveal that ZCWPW1 acts independently of PRDM9 as a chromatin-based intranuclear regulator of telomere architecture and telomere-led chromosome movements, thereby linking telomeric chromatin state to nuclear force transmission required for faithful meiotic progression. Significance StatementMeiotic pairing requires recombination and telomere-led chromosome movements, yet no chromatin factor has been shown to regulate both. We identify ZCWPW1 as the first chromatin-based regulator of rapid prophase movements. ZCWPW1 organizes telomeric chromatin and promotes cohesin and motor assembly at telomeres that for force transmission across the nuclear envelope. Loss of ZCWPW1 disrupts the telomere-nuclear envelope mechanical coupling, impairing motion and altering recombination. This function doesnt rely on PRDM9 despite their co-evolution and co-expression, challenging the prevailing view that ZCWPW1 only acts downstream of PRDM9 in DNA repair. Our findings show that chromatin readers can function as structural regulators of genome organization, revealing a conserved mechanism integrating chromosome structure, motion, and repair to ensure proper pairing and fertility.

cell biology↗

bioRxiv2024

Pentose Phosphate Pathway Protects E. coli from Antibiotic Lethality

Disruption of both branches of the canonical pentose phosphate pathway (PPP) in E. coli by combined inactivation of the zwf and talAB genes provokes the restoration of the ancient anabolic variant of PPP (aPPP). In the aPPP, pentose-5-phosphates are synthesized unidirectionally from fructose-6-phosphate and glyceraldehyde-3-phosphate by transketolase B, aldolase A, and phosphatase GlpX, converting sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate. Unexpectedly, the double zwf talAB mutant exhibits decreased survival after treatment by diverse classes of antibiotics with little effect on the minimal inhibitory concentration. Simultaneously, we found that killing effect of antimicrobials on the zwf talAB mutant could be reversed by the inactivation of either purR or deoB genes, both responsible for ribose-5-phosphate content in the mutant strain. Enhanced biosynthesis of the cell wall component ADP-heptose from sedoheptulose-7-phosphate also suppressed killing effect of antibiotics on the zwf talAB mutant. Furthermore, the inactivation of the Entner-Doudoroff pathway ({Delta}edd) or shifting the metabolic equilibrium by the addition of exogenous phosphogluconate reverts aPPP to glycolysis, preventing the accumulation of excess pentose phosphates and the occurrence of the futile cycle in zwf talAB cells, thus desensitizing them to antibiotics. Our findings show that ribose-5-phosphate metabolism plays a crucial role in bacterial tolerance to a wide range of bactericidal antibiotics. We propose that targeting PPP could be a promising strategy for developing new therapeutic agents aimed at potentiating clinically significant antimicrobials. IMPORTANCERecent studies have revealed the crucial role of bacterial cells metabolic status in its susceptibility to the lethal action of antibacterial drugs. However, there is still no clear understanding of which key metabolic nodes are optimal targets to improve the effectiveness of bacterial infection treatment. Our study establishes that the disruption of the canonical pentose phosphate pathway induces one-way anabolic synthesis of pentose phosphates (aPPP) in E. coli cells, significantly increasing the killing efficiency of various antibiotics. It is also demonstrated that the activation of ribose-5-phosphate utilization processes restores bacterial tolerance to antibiotics. We consider the synthesis of ribose-5-phosphate to be one of the determining factors of bacterial cell stress resistance. Understanding bacterial metabolic pathways, particularly the aPPPs role in antibiotic sensitivity, offers insights for developing novel adjuvant therapeutic strategies to enhance antibiotic potency.

microbiology↗

bioRxiv2024

Unraveling cysteine deficiency-associated rapid weight loss

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, -ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.

physiology↗

bioRxiv2023

Persistence of backtracking by human RNA polymerase II

RNA polymerase II (pol II) can backtrack during transcription elongation, exposing the 3 end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome wide distribution of more persistent backtracking is unknown. Consequently, we developed a novel method to directly sequence the extruded, "backtracked" 3 RNA. Our data shows that pol II slides backwards more than 20 nucleotides in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where pol II pauses near promoters and intron-exon junctions, and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking has the potential to affect diverse gene expression programs.

molecular biology↗

bioRxiv2023

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

biochemistry↗

bioRxiv2023

Structural mechanism of HP1-dependent transcriptional repression and chromatin compaction

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1 and nucleosomes. We have elucidated the cryo-EM structure of an HP1 dimer bound to an H2A.Z nucleosome, revealing that the HP1 dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1 binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1 functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

biochemistry↗

bioRxiv2023

General Transcription Factor from E. coli with a Distinct Mechanism of Action

Gene expression in E. coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) {sigma}70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of {beta} and {sigma}70 subunits to stabilize an open-promoter complex. Remarkably, CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress, and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis. One sentence summaryAn integrated structure-function approach uncovers CedA as a general transcription initiation factor in E. coli and elucidates its multifaceted role and unique mechanism.

molecular biology↗

bioRxiv2023

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1

The intricate regulation of chromatin plays a key role in controlling genome architecture and accessibility. Histone lysine methyltransferases regulate chromatin by catalyzing the methylation of specific histone residues but are also hypothesized to have equally important non-catalytic roles. SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation, and is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes suggesting the enzyme likely has uncharacterized non-catalytic activities. To characterize the catalytic and non-catalytic mechanisms SUV420H1 uses to modify chromatin, we determined cryo- EM structures of SUV420H1 complexes with nucleosomes containing histone H2A or its variant H2A.Z. Our structural, biochemical, biophysical, and cellular analyses reveal how both SUV420H1 recognizes its substrate and H2A.Z stimulates its activity, and show that SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from histone octamer. We hypothesize that this detachment increases DNA accessibility to large macromolecular complexes, a prerequisite for DNA replication and repair. We also show that SUV420H1 can promote chromatin condensates, another non-catalytic role that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

biochemistry↗

bioRxiv2023

Structural basis of histone H2A lysine 119 deubiquitination by Polycomb Repressive Deubiquitinase BAP1/ASXL1

The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. One Sentence SummaryWe reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

biochemistry↗

bioRxiv2022

Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity

Human shelterin is a six-subunit complex - comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1 - that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinantly produced components. By combining negative-stain electron microscopy (EM), crosslinking mass spectrometry (XLMS), mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they are extensively conformationally heterogenous. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 OB fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Both shelterin and the TRF1/TIN2/TPP1/POT1 subcomplex primarily adopt fully dimeric complexes, even in the absence of DNA substrates. TRF1/TIN2/TPP1/POT1 and shelterin complex showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

biochemistry↗

bioRxiv2021

Cryo-EM structure of the human CST·Polα/Primase complex in a recruitment state

The CST*Pol/Primase complex is essential for telomere overhang maintenance and additionally functions to counteract resection at double-strand breaks. We report a 4.6-[A] resolution cryo-EM structure of CST*Pol/Primase, captured prior to catalysis in a recruitment state, which provides insights into the architecture and stoichiometry of the fill-in machinery. Our model informs on human disease mutations that cause Coats plus syndrome.

biochemistry↗

bioRxiv2021

Inheritance of Repressed Chromatin Domains during S-phase Requires the Histone Chaperone NPM1

The epigenetic process safeguards cell identity during cell division through the inheritance of appropriate gene expression profiles. We demonstrated previously that parental nucleosomes are inherited by the same chromatin domains during DNA replication only in the case of repressed chromatin. We now show that this specificity is conveyed by NPM1, a histone H3/H4 chaperone. Proteomic analyses of late S-phase chromatin revealed NPM1 in association with both H3K27me3, an integral component of facultative heterochromatin and MCM2, an integral component of the DNA replication machinery; moreover NPM1 interacts directly with PRC2 and with MCM2. Given that NPM1 is essential, the inheritance of repressed chromatin domains was examined anew using mESCs expressing an auxin-degradable version of endogenous NPM1. Upon NPM1 degradation, cells accumulated in S-phase of the cell-cycle and parental nucleosome inheritance from repressed chromatin domains was markedly compromised. Appropriate inheritance required the NPM1 acidic patches that function in chaperone activity, pointing to NPM1 being integral to the epigenetic process. One-Sentence SummaryThe histone H3/H4 chaperone, NPM1, fosters epigenetic inheritance from parental repressed chromatin during DNA replication.

molecular biology↗

bioRxiv2021

Lost in translation: codon optimization inactivates SARS-CoV-2 RdRp

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors. HighlightsO_LICodon-optimization of Nsp12 triggers misfolding and activity loss C_LIO_LISlow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12 C_LIO_LINon-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domain C_LIO_LICrosstalk between two Nsp12 active sites that bind the same ligands C_LI

molecular biology↗

bioRxiv2018

YcjW: a transcription factor that controls the emergency generator of H2S in E. coli.

Hydrogen sulfide (H2S) is a ubiquitous gaseous molecule that is endogenously produced in both eukaryotes and prokaryotes. Its role as a pleiotropic signaling molecule has been well characterized in mammals1,2. In contrast, the physiological role of H2S in bacteria only recently became apparent; H2S acts as a cytoprotectant against antibiotics-induced stress and affect the cells ability to maintain redox homeostasis 3-5. In E. coli, endogenous H2S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA, previously known as sseA3,4. Here, we show that cells lacking 3MST acquired a unique phenotypic suppressor mutation resulting in compensatory H2S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we mapped a non-synonymous single nucleotide polymorphism (SNP) to uncharacterized Laci-type transcription factor, YcjW. We identified transcriptional regulatory targets of YcjW and discovered a major target, thiosulfate sulfurtransferase PspE, as an alternative mechanism for H2S biosynthesis. Deletion of pspE was sufficient to antagonize phenotypic suppression. Our results reveal a complex interaction between cell metabolism and H2S production and the role, a hithero uncharacterized transcription factor, YcjW, plays in linking the two.

microbiology↗

bioRxiv2018

Analysis of the impact of molecular motions on the efficiency of XL-MS and the distance restraints in hybrid structural biology

Covalent cross-link mapping by mass spectrometry (XL-MS) is rapidly becoming the most widely used method of hybrid structural biology. We investigated the impact of incremental variations of cross-linker length have on the depth of XL-MS interrogation of protein-protein complexes, and assessed the role molecular motions in solution play in generation of cross-link-derived distance restraints. Supplementation of a popular NHS-ester cross-linker, DSS, with 2 reagents shorter or longer by CH2-CH2, increased the number of non-reductant cross-links by ~50%. Molecular dynamics simulations of these cross-linkers revealed 3 individual, partially overlapping ranges of motion, consistent with partially overlapping sets of cross-links formed by each reagent. Similar simulations elucidated protein fold-specific ranges of motions for the reactive and backbone atoms from rigid and flexible target domains. Together these findings create a quantitative framework for generation of cross-linker- and protein fold-specific distance restraints for XL-MS-guided protein-protein docking.

molecular biology↗

bioRxiv2018

An automated workflow for the discovery and docking simulation of the protein-protein complexes using in vivo chemical cross-linking

Introduction Introduction Results Discussion Materials and Methods Competing interests Contributions Materials and Correspondence References Chemical cross(X)-link mapping assisted by mass spectrometry (XL-MS, also CXMS and CLMS) is a low-resolution hybrid method of structural biology, yielding a set of pairwise distance restraints between reactive solvent-accessible amino acids1-9. Most commonly used X-linkers at present belong to the class of amino-reactive homo-bifunctional NHS-esters, which act as protein proximity sensors, connecting predominantly Lys residues (as well as far less common and informative N-terminal amines)10-14. Main structural application of XL-MS to date i ...

molecular biology↗