About

Marc Greenberg is the Vernon K. Krieble Professor of Chemistry at Johns Hopkins University, where he has been a faculty member since 2002. His research group focuses on organic and bioorganic chemistry, chemical biology, with particular emphasis on nucleic acids. Greenberg's work involves using organic chemistry, biochemistry, and molecular biology to understand and exploit the reactivity and structure of nucleic acids, aiming to address fundamental questions about nucleic acid chemistry and develop practical tools for biotechnology. He received his Ph.D. at Yale University under Professor Jerome Berson and trained as an American Cancer Society Postdoctoral Fellow at Caltech in Professor Peter Dervan's laboratory. Greenberg holds undergraduate degrees in Chemistry from New York University and in Chemical Engineering from the Cooper Union School of Engineering. He is a Fellow of the American Association for the Advancement of Science and has received several awards, including the Arthur C. Cope Scholar Award from the American Chemical Society in 2016 and an Alfred P. Sloan Foundation fellowship from 1996 to 2000. His research explores the chemical biology of nucleic acids, including understanding how cytotoxic antitumor agents target DNA, and developing tools for biotechnology. His group synthesizes novel molecules and studies their behavior using various physicochemical, biochemical, and biological techniques. Greenberg has also served as the Founding Director of the Chemistry-Biology Interface program at Johns Hopkins University.

Research topics

• Chemistry
• Biochemistry
• Stereochemistry
• Molecular biology
• Photochemistry

Selected publications

• Protein–Protein Cross-Linking by a DNA Damage-Derived Histone Modification

  Journal of the American Chemical Society · 2026-04-17

  articleSenior authorCorresponding

  Nonenzymatic covalent modification (NECM) of lysine residues can be physiologically consequential. A NECM is formed by the oxidized abasic site (C4-AP), which is produced by DNA-damaging agents. C4-AP reacts with the ε-amine of histone lysines in nucleosome core particles (NCPs) to form an electrophilic 5-methylene pyrrolone NECM (KMP). KMP is also produced on histones in bleomycin-treated human cells. Here, we describe a molecule (1a) that yields KMP by reacting directly with histones in NCPs. KMP forms on lysines of all four core histones in the order H3 > H2A/H2B > H4. Biotinylated KMP-containing NCPs prepared using 1a were incubated with HeLa nuclear lysates in the presence of glutathione. NCP–protein cross-links were observed by native PAGE. Protein–protein cross-links (PPCs) were enriched through intact NCP pull-down and identified via tryptic digests by LC-MS/MS. Model reactions demonstrate KMP is more electrophilic than N-acyllysine post-translational modifications (PTMs) but does not form PPCs indiscriminately within NCPs. Enriched proteins are functionally biased, with overrepresentation of DNA binding, histone binding, histone PTMs, and transcription regulation. Proteins enriched by KMP-containing NCPs produced by generating C4-AP on DNA were analyzed in parallel. Similar overrepresented functions were observed when C4-AP was introduced near the H3/H4 N-tails, whereas a distinct group of proteins was enriched when C4-AP was introduced near the H2A acidic patch. PPC formation by KMP is modulated by the NCP environment. Combined with the known intracellular formation of KMP, this study inspires investigating whether PPC formation by this NECM impacts cell function and viability.

  PublisherDOI

• Noncompetitive Inhibition of DNA Polymerase β by a Nonnative Nucleotide

  The Journal of Organic Chemistry · 2025-09-07

  articleOpen accessSenior authorCorresponding

  Base excision repair (BER) is a DNA repair pathway responsible for protecting the genome against modified nucleotides. DNA polymerase β (Pol β) participates in this process by removing the remnants of a damaged nucleotide and filling in the resulting gap. Pol β is overexpressed in some cancers and is synthetic lethal in cells deficient in BRCA1/2, providing additional impetus for identifying inhibitors of this enzyme. We report noncovalent Pol β inhibitors that are nonnative nucleotides. The inhibitors were identified via a combination of structural and biochemical analysis, as well as serendipity, from an initial library of covalent inhibitor candidates in which diversity was introduced sequentially at the C3′- and C5-positions of pyrimidine nucleotides. The molecules are among the most potent Pol β inhibitors (Ki ≤ 70 nM) of the enzyme’s polymerase and lyase activities. Kinetic analyses reveal that the molecules inhibit Pol β noncompetitively. Fluorescence anisotropy and kinetic experiments reveal that the more potent inhibitor binds in the lyase domain and does not prevent DNA binding. Neither the more potent noncompetitive inhibitor nor a neutral protide exhibits cytotoxic synergism with the DNA damaging agent methyl methanesulfonate in HeLa cells. Cell permeability experiments suggest that micromolar levels of the more potent noncompetitive inhibitor and corresponding protide are taken up by HeLa cells following 24 h incubation (25 μM). However, based upon a comparison with other molecules, it is possible that they are membrane bound. The molecules identified could be useful tools in biochemical studies and provide a starting point for creating new Pol β inhibitors that function in cells.

  PublisherOA PDFDOI

• Inhibitors of SAMHD1 Obtained from Chemical Tethering to the Guanine Antiviral Acyclovir

  Biochemistry · 2025-02-24 · 3 citations

  articleOpen accessCorresponding

  Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is an enzyme with diverse activities. Its dNTPase activity degrades all canonical dNTPs and many anticancer nucleoside drugs, while its single-stranded nucleic acid binding activity promotes DNA repair and RNA homeostasis in cells. These functions require guanine nucleotide binding to a specific allosteric site (A1) on the enzyme. We previously described how the activities of SAMHD1 could be inhibited in vitro with fragment-based inhibitor design, using dGMP as a targeting fragment for the A1 site. However, these dGMP-tethered inhibitors had poor cell permeability due to the charged guanine monophosphate group. Here, we describe a new approach where the amino form of the guanine acyclic nucleoside acyclovir (NH2-ACV) is used as the targeting fragment, allowing facile coupling to activated carboxylic acids (R–COOH), either directly or using linkers. This approach generates a neutral amide instead of charged monophosphate attachment points. High-throughput screening of a ∼375 compound carboxylic acid library identified two compounds (8, 11) with similar micromolar affinities for SAMHD1. Compound 11 was obtained by direct coupling to NH2-ACV, while compound 8 used a five-carbon linker. Both inhibitors had the same dibromonaphthol component from the carboxylic acid library screen. A crystal structure of a complex between SAMHD1 and 8, combined with computational models of bound 11, suggest how the dibromonaphthol promotes binding. The findings establish that guanine-based inhibitors targeting the A1 site do not require nucleotide or cyclic nucleoside structural elements. This guanine site targeting strategy is highly amenable to further chemical optimization.

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• Optimizing bone health for the prevention of revision adult spinal deformity surgery: a break-even analysis

  Spine Deformity · 2025-04-11

  article
  PublisherDOI

• Uridine Embedded within DNA is Repaired by Uracil DNA Glycosylase via a Mechanism Distinct from That of Ribonuclease H2

  Journal of the American Chemical Society · 2025-03-25 · 8 citations

  articleOpen accessCorresponding

  Uridine (rU) and 2′-deoxyuridine (dU) are common DNA lesions. dU is repaired through a base excision repair (BER) pathway initiated by uracil DNA glycosylase (UDG), while rU is typically removed from DNA via ribonucleotide excision repair, mediated by RNase H2. In this study, we report that rU is also repaired through the UDG-mediated BER pathway. We found that UDG catalyzes the removal of uracil from rU embedded in DNA, but exhibits no activity toward rU in RNA. Biochemical and crystallographic analyses revealed that the 2′–OH group of rU is effectively accommodated by UDG and directly participates in catalyzing the hydrolysis of the N-glycosidic bond. The abasic site product generated upon removal of uracil from rU by UDG is further processed by downstream BER enzymes to restore undamaged DNA. Our findings suggest that UDG-initiated BER constitutes a previously unrecognized pathway for the repair of rU-specific ribonucleotides. Additionally, we developed a method for selectively quantifying rU content in DNA. Using this method, we determined that rU repair by UDG is not a major pathway in human cells. This discovery expands our understanding of the diverse biological functions of UDG, and inspires further investigation to determine the role of its rU-removal in cells.

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• A human high-fidelity DNA polymerase holoenzyme has a wide range of lesion bypass activities

  Nucleic Acids Research · 2025-07-08 · 1 citations

  articleOpen access

  During replication, lagging strand lesions are initially encountered by high-fidelity DNA polymerase (pol) holoenzymes comprised of pol δ and the PCNA sliding clamp. To proceed unhindered, pol δ holoenzymes must bypass lesions without stalling. This entails dNMP incorporation opposite the lesion (insertion) and the 5' template deoxynucleotide (extension). Historically, it was viewed that high-fidelity pol holoenzymes stall upon encountering lesions, activating DNA damage tolerance pathways that are ultimately responsible for lesion bypass. Our recent study of four prominent lesions revealed that human pol δ holoenzymes support insertion and/or bypass for multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. In the present study, we expand these analyses to other prominent lesions. Collectively, analyses of 10 lesions from both studies reveal that the insertion and bypass efficiencies of pol δ holoenzymes each span a complete range (0%-100%). Consequently, the fates of pol δ holoenzymes upon encountering lesions are quite diverse. Furthermore, pol δ proofreading promoted holoenzyme progression at 7 of the 10 lesions and did not deter progression at any. Altogether, the results significantly alter our understanding of the replicative capacity of high-fidelity pol holoenzymes and their functional role(s) in lesion bypass.

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• Examination of a Chimeric Bis-Electrophile for Selective DNA–Protein Cross-Linking and Mechlorethamine Reveals an Unknown Source of Nitrogen Mustard Cytotoxicity

  Journal of the American Chemical Society · 2025-08-19 · 1 citations

  articleOpen accessSenior authorCorresponding

  DNA–protein cross-links (DPCs) are cytotoxic lesions whose study in cells is complicated by the lack of exogenous agents that produce them selectively over DNA–DNA interstrand cross-links (ICLs). The synthesis and reactivity of a chimeric bis-electrophile (MEBAC) that is comprised of a highly reactive alkylating agent and a lysine selective o-ethynyl benzaldehyde is described. DPC formation in nucleosome core particles (NCPs) by MEBAC is >40-times greater than that of ICLs. Cell viability experiments and the single cell Comet assay are consistent with NCP reactivity. Compared to a nitrogen mustard (mechlorethamine, MCE) MEBAC produces higher DPC yields and lower ICL yields in NCPs and in cells at comparable cytotoxicity. Cell viability experiments show that while DPCs from MEBAC are repaired by the metalloprotease SPRTN and the proteasome, only the former repairs such lesions produced by MCE. The inability of the proteasome to repair DPCs in MCE-treated cells likely contributes to the cytotoxicity of the nitrogen mustard. Proteomic analysis identifies several cysteine-rich E3 ligases involved in ubiquitination that are cross-linked to DNA in MCE-treated but not MEBAC-treated cells and suggests a chemical basis for why DPCs produced by the nitrogen mustard are not repaired by the proteasome. This investigation reveals a previously unknown source of nitrogen mustard cytotoxicity and indicates that MEBAC and molecules like it will be useful tools for studying DPCs in cells.

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• Biochemical and structural characterization of Fapy•dG replication by Human DNA polymerase β

  Nucleic Acids Research · 2024-04-08 · 3 citations

  articleOpen accessSenior author

  N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase β (Pol β), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol β incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

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• 8-OxodGuo and Fapy•dG Mutagenicity in <i>Escherichia coli</i> Increases Significantly when They Are Part of a Tandem Lesion with 5-Formyl-2′-deoxyuridine

  Chemical Research in Toxicology · 2024-07-23 · 2 citations

  articleOpen access

  Tandem lesions, which are defined by two or more contiguously damaged nucleotides, are a hallmark of ionizing radiation. Recently, tandem lesions containing 5-formyl-2′-deoxyuridine (5-fdU) flanked by a 5′-8-OxodGuo or Fapy•dG were discovered, and they are more mutagenic in human cells than the isolated lesions. In the current study, we examined replication of these tandem lesions in Escherichia coli. Bypass efficiency of both tandem lesions was reduced by 30–40% compared to the isolated lesions. Mutation frequencies (MFs) of isolated 8-OxodGuo and Fapy•dG were low, and no mutants were isolated from replication of a 5-fdU construct. The types of mutations from 8-OxodGuo were targeted G → T transversion, whereas Fapy•dG predominantly gave G → T and G deletion. 5′-8-OxodGuo-5-fdU also gave exclusively G → T mutation, which was 3-fold and 11-fold greater, without and with SOS induction, respectively, compared to that of an isolated 8-OxodGuo. In mutY/mutM cells, the MF of 8-OxodGuo and 5′-8-OxodGuo-5-fdU increased 13-fold and 7-fold, respectively. The MF of 5′-8-OxodGuo-5-fdU increased 2-fold and 3-fold in Pol II- and Pol IV-deficient cells, respectively, suggesting that these polymerases carry out largely error-free bypass. The MF of 5′- Fapy•dG-5-fdU was similar without (13 ± 1%) and with (16 ± 2%) SOS induction. Unlike the complex mutation spectrum reported earlier in human cells for 5′- Fapy•dG-5-fdU, with G → T as the major type of errors, in E. coli, the mutations were predominantly from deletion of 5-fdU. We postulate that removal of adenine-incorporated opposite 8-OxodGuo by Fpg and MutY repair proteins is partially impaired in the tandem 5′-8-OxodGuo-5-fdU, resulting in an increase in the G → T mutations, whereas a slippage mechanism may be operating in the 5′- Fapy•dG-5-fdU mutagenesis. This study showed that not only are these tandem lesions more mutagenic than the isolated lesions but they may also exhibit different types of mutations in different organisms.

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• Quantification of Intracellular DNA–Protein Cross-Links with N7-Methyl-2′-Deoxyguanosine and Their Contribution to Cytotoxicity

  Chemical Research in Toxicology · 2024-04-23 · 4 citations

  articleOpen accessSenior authorCorresponding

  The major product of DNA-methylating agents, N7-methyl-2′-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA–protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

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Recent grants

• NIH Grant R01GM063028

  NIH · $5.4M · 2020

• NIH Grant R01CA074954

  NIH · $1.7M · 2008

• NIH Grant R01ES027558

  NIH · $1.7M · 2024

• Selective Detection and Quantification of DNA Lesions

  NSF · $480k · 2010–2014

• NIH Grant R29GM046534

  NIH · $502k · 1997

Frequent coauthors

• Kelly M. Kroeger

  Agilent Technologies (United States)

  31 shared

• Brian C. Bales

  GE Global Research (United States)

  27 shared

• Carissa J. Wiederholt

  Johns Hopkins University

  25 shared

• Michael O. Delaney

  Cornell University

  22 shared

• Myron F. Goodman

  University of Southern California

  21 shared

• Murat Saparbaev

  Institut Gustave Roussy

  20 shared

• Jacques Laval

  Université Paris-Saclay

  20 shared

• Jaeseung Kim

  Qurient (South Korea)

  20 shared

Labs

• Marc GreenbergPI

Awards & honors

• Arthur C. Cope Scholar Award from the American Chemical Soci…
• Fellow of the American Association for the Advancement of Sc…
• Alfred P. Sloan Foundation fellow (1996-2000)
• Founding Director of the Chemistry-Biology Interface program…