Stephen Benkovic

Extrachromosomal DNA (ecDNA) presents a major challenge for precision medicine, contributing to poor survival for patients with oncogene-amplified tumours. EcDNA renders tumours resistant to targeted treatments by facilitating massive transcription of oncogenes and rapid genome evolution. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers, exposing an actionable vulnerability. Stepwise analyses of ecDNA transcription reveal landscapes of pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation onto growing DNA strands is markedly slower on ecDNA, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. Replication Protein A2 phosphorylated on serine 33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours as they enter S-phase. To exploit this vulnerability, we develop a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, and demonstrate that it preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing …

Aims Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH. Methods and results Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in …

The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 …

Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid …

Background Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis after angioplasty or stent placement. VSMCs reprogram their metabolism to meet the increased requirements of lipids, proteins, and nucleotides for their proliferation. De novo purine synthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined. Methods De novo purine synthesis in proliferative VSMCs was evaluated by liquid chromatography-tandem mass spectrometry. The expression of ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last 2 steps of the de novo purine synthesis pathway, was assessed in VSMCs of proliferative arterial neointima. Global and VSMC-specific knockout of …

Over the past fifteen years, we have unveiled a new mechanism by which cells achieve greater efficiency in de novo purine biosynthesis. This mechanism relies on the compartmentalization of de novo purine biosynthetic enzymes into a dynamic complex called the purinosome. In this review, we highlight our current understanding of the purinosome with emphasis on its biophysical properties and function and on the cellular mechanisms that regulate its assembly. We propose a model for functional purinosomes in which they consist of at least ten enzymes that localize near mitochondria and carry out de novo purine biosynthesis by metabolic channeling. We conclude by discussing challenges and opportunities associated with studying the purinosome and analogous metabolons.

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein–protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB …

The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical properties. Early literature evidence suggested that they might assemble into a multi-enzyme complex called a metabolon. The finding that fluorescently-tagged chimeras of the pathway enzymes form discrete puncta, now called purinosomes, is further elaborated in this review to include: a discussion of their assembly; the role of ancillary proteins; their locus at the microtubule/mitochondria interface; the elucidation that at endogenous levels, purinosomes function to channel intermediates from phosphoribosyl pyrophosphate to AMP and GMP; and the evidence for the purinosomes to exist as a protein condensate. The review concludes with a consideration of probable signaling pathways that might promote the assembly and disassembly of the …

With the elucidation of myriad anabolic and catabolic enzyme-catalyzed cellular pathways crisscrossing each other, an obvious question arose: how could these networks operate with maximal catalytic efficiency and minimal interference? A logical answer was the postulate of metabolic channeling, which in its simplest embodiment assumes that the product generated by one enzyme passes directly to a second without diffusion into the surrounding medium. This tight coupling of activities might increase a pathway's metabolic flux and/or serve to sequester unstable/toxic/reactive intermediates as well as prevent their access to other networks. Here, we present evidence for this concept, commencing with enzymes that feature a physical molecular tunnel, to multi-enzyme complexes that retain pathway substrates through electrostatics or enclosures, and finally to metabolons that feature collections of enzymes …

Early Life and Education Tom was born in Los Angeles on August 25, 1925. His father left the family three years later. His mother, who had only a sixth-grade education, had to put Tom in foster care. When he was eight, he went to live at the McKinley Home for Boys, a facility in Van Nuys for 200 boys who either had no parents or had parents unable to take care of them. There he attended school for the first time. During his four years at McKinley, hisThomas C. Bruice was one of the fathers of bioorganic chemistry. His productivity was prodigious, as was his creativity, which led to more than 600 papers whose wealth of discovery contributed to a foundation for the development of bioorganic chemistry. His papers fell broadly into several distinct categories: How do enzymes achieve their enormous catalytic rate advantages relative to a nonenzymatic reference state? What are the mechanisms used by various coenzymes to catalyze specific transformations? How are general acid-base catalysis and transition-state stabilization harnessed by enzymes to facilitate various reactions?

Replication: DNA replication fork, bacterial — Penn State Skip to main navigation Skip to search Skip to main content Penn State Home Penn State Logo Help & FAQ Home Researchers Research output Research units Equipment Grants & Projects Prizes Activities Search by expertise, name or affiliation Replication: DNA replication fork, bacterial MM Spiering, SJ Benkovic Chemistry Research output: Chapter in Book/Report/Conference proceeding › Chapter Overview Original language English (US) Title of host publication Encyclopedia of Biological Chemistry Subtitle of host publication Third Edition Publisher Elsevier Pages 49-53 Number of pages 5 Volume 4 ISBN (Electronic) 9780128220405 ISBN (Print) 9780128194607 DOIs https://doi.org/10.1016/B978-0-12-819460-7.00551-X State Published - Jul 29 2021 All Science Journal Classification (ASJC) codes Biochemistry, Genetics and Molecular Biology(all) …

2015-08-20 First worldwide family litigation filed litigation Critical https://patents. darts-ip. com/? family= 38225302&utm_source= google_patent&utm_medium= platform_link&utm_campaign= public_patent_search&patent= BRPI0621279 (B1)" Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.

I endeavor to share how various choices—some deliberate, some unconscious—and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

A method is disclosed for altering the redox equilibrium of a transmembrane receptor-like protein tyrosine phosphatase (RPTP), particularly a type IIa RPTP, by chemically modu lating the activity through contact of such an enzyme with a diaryl boron compound that preferentially reacts with anRPTP in an oxidized state (CyS-OH). This redox modulation is most readily observed and put to use by assaying the inhibition of the phosphatase activity of the RPTP. Such an assay can be carried out on an enzyme in vitro or by contacting the enzyme in a living organism (in vivo). A useful diaryl boron compound corresponds in structure to

Dataset supporting the paper: Doigneaux, C., Pedley, A., Mistry, I. N., Papayova, M., Benkovic, S., & Tavassoli, A. (Accepted/In press). Hypoxia drives the assembly of the multi-enzyme purinosome complex. The Journal of Biological Chemistry. DOI:10.1074/jbc.RA119.012175

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic “hotspots” within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

Efficient de novo purine biosynthesis in higher eukaryotes is hypothesized to depend on the existence of a multienzyme complex — the purinosome. However, there is as yet no compelling identification of specific protein‐protein interactions between purine biosynthetic enzymes. Similarly, functionally important interactions between purine biosynthetic enzymes and other proteins have yet to be elucidated. Inspired by the highly symmetric oligomeric structure of PAICS, a de novo purine biosynthetic enzyme, we identified several putative interaction surfaces. Using genetically encoded unnatural amino acid incorporation, we inserted photocrosslinking amino acid analogs into these surfaces at specific residues. Upon UV irradiation, we were able to detect multiple crosslinked products between PAICS and other proteins, formed in their native context inside living cells. Distinct sites of photocrosslinker incorporation …

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates …

Inhibitors of the tmRNA pathway have antibacterial activity with broad species specificity, including B. anthracis and other pathogens of military and civilian interest. Identified cyclic or linear peptides are further selected by in vivo selection methods, kill bacterial pathogens when added exogenously, and/or eliminate plasmids carrying antibiotic resistance or virulence genes. The molecular target of each cyclic peptide is in the tmRNA pathway and the mRNA pathway is inhibited in vitro and in vive by the addition of the peptides.

Paintings often consist of highly complex layered structures that contain a mixture of organic and inorganic materials at each layer depending upon the artist’s technique. A comprehensive analysis of the chemical composition could provide critical information on the sequence of events that led to an artwork’s current state. In this investigation, we have employed a novel imaging technique known as Secondary Ion Mass Spectrometry (SIMS) to chemically map the cross-section of a paint film sampled from Leonardo da Vinci’s Adoration of the Magi at a submicron resolution using a C60+ ion source. A wide range of materials were found to be spatially localized at the different layers such as: protein, carbon, silicates, fatty acids, salts and lead associated compounds. An interaction of chloride salts with the lead white was observed in the priming layer. The chloride salts disrupted most of the priming layer suggesting that a cleaning process took place that removed most of the priming layer before the monochrome brown repaints were added on top at a later time.

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Tel:(706) 713-2216; Fax:(706) 713-2221; E-mail; ude. agu@ thgualsc, or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.

The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we …