Formation Of DNA-protein Cross-links Research Articles

Endogenous Cellular Metabolite Methylglyoxal Induces DNA-Protein Cross-Links in Living Cells.

Methylglyoxal (MGO) is an electrophilic α-oxoaldehyde generated endogenously through metabolism of carbohydrates and exogenously due to autoxidation of sugars, degradation of lipids, and fermentation during food and drink processing. MGO can react with nucleophilic sites within proteins and DNA to form covalent adducts. MGO-induced advanced glycation end-products such as protein and DNA adducts are thought to be involved in oxidative stress, inflammation, diabetes, cancer, renal failure, and neurodegenerative diseases. Additionally, MGO has been hypothesized to form toxic DNA-protein cross-links (DPC), but the identities of proteins participating in such cross-linking in cells have not been determined. In the present work, we quantified DPC formation in human cells exposed to MGO and identified proteins trapped on DNA upon MGO exposure using mass spectrometry-based proteomics. A total of 265 proteins were found to participate in MGO-derived DPC formation including gene products engaged in telomere organization, nucleosome assembly, and gene expression. In vitro experiments confirmed DPC formation between DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as histone proteins H3.1 and H4. Collectively, our study provides the first evidence for MGO-mediated DNA-protein cross-linking in living cells, prompting future studies regarding the relevance of these toxic lesions in cancer, diabetes, and other diseases linked to elevated MGO levels.

Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB

Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Abstract 7113: Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents

Abstract Bifunctional alkylating agents, such as nitrosoureas, platinum-based therapeutics, and chloroethylhydrazines, have demonstrated clinical successes against cancer by inducing cytotoxic interstrand or intrastrand DNA crosslinks. However, the less well-characterized capacity of these agents to couple DNA to chromosomally-associated proteins may also contribute to these compounds’ observed cytotoxicities. Bulky DNA-protein crosslinks (DPCs) could obstruct the DNA metabolism critical to cancer proliferation, thus contribute to genomic instability and cell death. To endogenously repair DPCs, human cells often utilize a DPC-specific proteolytic mechanism dependent on the metalloprotease SPRTN. Complementary approaches for quantifying DPCs, generated ex vivo, to assess the capacity of several bifunctional alkylating agents to induce these lesions in cultured human leukemia cells are presented here. In the first approach, proteins were isolated from lysates of drug-treated cells and protein-bound DNA was quantified as a ratio to the total DNA present in the lysates. In the complementary approach, DNA was extracted from lysates of drug-treated cells using a modified ‘STAR’ assay in which genomic DNA is isolated and the DNA-bound proteins were quantified by fluorescein isothiocyanate labeling. Micromolar concentrations of the nitrogen mustard mechlorethamine and nitrosourea lomustine induced significant DPC formation while more modest DPC formation was observed using similar concentrations of the chloroethylating agents carmustine and laromustine, as well as cisplatin. Bifunctional alkylating agents dependent on epoxide functionality, including dianhydrogalacticol and diepoxybutane, required drug concentrations of at least one order of magnitude larger to produce similar DPC yields measurable by these techniques. A strategy to interfere with the DPC repair pathway via SPRTN knockdown and assess increased sensitivity of cancer cells to bifunctional alkylating agents is also presented. Chemotherapeutic sensitization by targeting SPRTN could suggest a therapeutic strategy to improve effectiveness of several DNA-targeted anticancer agents, especially if extended to other therapeutics that trap DPCs such as inhibitors of topoisomerase and poly(ADP-ribose) polymerase. Further, identifying structure-function relationships with respect to bifunctional alkylating agents and DPC formation could inform the future development and clinical utilization of these chemotherapies. Citation Format: Cole Turner, Hailey Cerrato, Victoria Melehov, Charlotte Healy, Kevin P. Rice. Targeting the repair of DNA-protein crosslinks induced by bifunctional alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7113.

Mass Spectrometry Evidence for Forming Schiff Base 3'-DNA-Histone Cross-Links from Abasic Sites in Vitro and in Human Cells.

Histones catalyze DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by the formation of reversible but long-lived DNA-protein cross-links at 3'-termini (3'-histone-DPCs). However, the chemical structures of 3'-histone-DPCs are not well characterized, and whether they are formed in cells is uncertain. In this study, we developed a liquid chromatography with tandem mass spectrometry workflow to characterize DPCs produced from the reaction of histones with AP sites and wish to report evidence that histones cross-link to incised AP sites via Schiff bases. We also demonstrated for the first time that 3'-histone-DPCs are produced endogenously in human embryonic kidney 293T cells.

DNA-Histone Cross-Link Formation via Hole Trapping in Nucleosome Core Particles.

Radical cations (holes) produced in DNA by ionizing radiation and other oxidants yield DNA-protein cross-links (DPCs). Detailed studies of DPC formation in chromatin via this process are lacking. We describe here a comprehensive examination of DPC formation within nucleosome core particles (NCPs), which are the monomeric component of chromatin. DNA holes are introduced at defined sites within NCPs that are constructed from the bottom-up. DPCs form at DNA holes in yields comparable to those of alkali-labile DNA lesions that result from water trapping. DPC-forming efficiency and site preference within the NCP are dependent on translational and rotational positioning. Mass spectrometry and the use of mutant histones reveal that lysine residues in histone N-terminal tails and amino termini are responsible for the DPC formation. These studies are corroborated by computational simulation at the microsecond time scale, showing a wide range of interactions that can precede DPC formation. Three consecutive dGs, which are pervasive in the human genome, including G-quadruplex-forming sequences, are sufficient to produce DPCs that could impact gene expression.

Synthesis and Excision Repair of Site-Specific 3'-End DNA-Histone Cross-Links Derived from Abasic Sites.

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

N-Acyloxymethyl-phthalimides deliver genotoxic formaldehyde to human cells.

Formaldehyde is a pollutant and human metabolite that is toxic at high concentrations. Biological studies on formaldehyde are hindered by its high reactivity and volatility, which make it challenging to deliver quantitatively to cells. Here, we describe the development and validation of a set of N-acyloxymethyl-phthalimides as cell-relevant formaldehyde delivery agents. These esterase-sensitive compounds were similarly or less inhibitory to human cancer cell growth than free formaldehyde but the lead compound increased intracellular formaldehyde concentrations, increased cellular levels of thymidine derivatives (implying increased formaldehyde-mediated carbon metabolism), induced formation of cellular DNA-protein cross-links and induced cell death in pancreatic cancer cells. Overall, our N-acyloxymethyl-phthalimides and control compounds provide an accessible and broadly applicable chemical toolkit for formaldehyde biological research and have potential as cancer therapeutics.

DNA-protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM).

In higher eukaryotic cells, mitochondria are essential organelles for energy production, metabolism, and signaling. Mitochondrial DNA (mtDNA) encodes 13 protein subunits for oxidative phosphorylation and a set of tRNAs and rRNAs. mtDNA damage, sourced from endogenous chemicals and environmental factors, contributes to mitochondrial genomic instability, which has been associated with various mitochondrial diseases. DNA-protein cross-links (DPCs) are deleterious DNA lesions that threaten genomic integrity. Although much has been learned about the formation and repair of DPCs in the nucleus, little is known about DPCs in mitochondria. Here, we present in vitro and in cellulo data to demonstrate the formation of DPCs between a prevalent abasic (AP) DNA lesion and a DNA-packaging protein, mitochondrial transcription factor A (TFAM). TFAM cleaves AP-DNA and forms DPCs and single-strand breaks (SSB). Lys residues of TFAM are critical for the formation of TFAM-DPC and a reactive 3'-phospho-α,β-unsaturated aldehyde (3'pUA) residue on SSB. The 3'pUA residue reacts with two Cys of TFAM and contributes to the stable TFAM-DPC formation. Glutathione reacts with 3'pUA and competes with TFAM-DPC formation, corroborating our cellular experiments showing the accumulation of TFAM-DPCs under limiting glutathione. Our data point to the involvement of TFAM in AP-DNA turnover and fill a knowledge gap regarding the protein factors in processing damaged mtDNA.

An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.

DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

Bactericidal effects of plasma‐activated saline prepared by surface dielectric barrier discharge with different dielectric layers and working gases

AbstractThe polytetrafluoroethylene (PTFE) serves as a dielectric layer for surface dielectric barrier discharge (SDBD) and the prepared plasma‐activated saline (PAS) exhibited strong bactericidal effects, however, the fluorides are inevitably generated and bring about side effects in biological application. In this study, the SDBD devices with the dielectric layers of quartz or ceramic compared with that with PTFE were utilized to explore the effective and fluoride‐free PAS using different working gases. The Quartz‐PAS prepared with air exhibited a relatively strong bactericidal effect without inducing the DNA‐protein crosslinks (DPCs) in Escherichia coli cells, while the Ceramic‐PAS exhibited a weak bactericidal effect and induced the DPCs formation. This study supplies guidance in the preparation and regulation of PAS for biomedical applications.

The SOS response-associated peptidase (SRAP) domain of YedK catalyzes ring opening of abasic sites and reversal of its DNA–protein cross-link

Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA–protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3′-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.

DNA-Protein Crosslinks and Their Resolution.

Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans.

Free-Radical-Mediated Photoinduced Electron Transfer between 6-Thioguanine and Tryptophan Leading to DNA-Protein-Like Cross-Link.

The nucleobase analog 6-thioguanine (6-TG) has emerged as important immunosuppressant, anti-inflammatory, and anticancer drug in the past few decades, but its unique photosensitivity of absorbing strongly ultraviolet UVA light elicits photochemical hazards in many ways. The particularly intriguing yet unresolved question is whether the direct photoreaction of 6-TG can promote DNA-protein cross-links (DPCs) formation, which are large DNA adducts blocking DNA replication and physically impede DNA-related processes. Herein, by real-time observation of radical intermediates using time-resolved UV-vis absorption spectroscopy in conjunction with product analysis by HPLC-MS, we discover that UVA excitation of 6-TG triggers direct covalent cross-linking with tryptophan (TrpH) via an exquisite radical mechanism of electron transfer. The photoexcitation prepares the redox-active triplet 36-TG*, which initiates electron transfer with TrpH, creating TrpH•+ and 6-TG•- in the first step. The deprotonated Trp• undergoes radical-recombination with its geminate partner 6-TG•- and eliminates a H2S, leading to the cross-linking product 6-TG-Trp. The photoadduct structures (two chiral isomers and one constitutional isomer) are identified unambiguously, validating further the mechanism. These findings pinpoint the exact amino acid that is vulnerable to photo-cross-linking with 6-TG and establish a mechanistic framework for understanding mutagenic DPCs formation and developing photoprobes based on this new type of photo-cross-linking.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

Astragalus polysaccharide protects formaldehyde-induced toxicity by promoting NER pathway in bone marrow mesenchymal stem cells.

In our previous study, it has been confirmed that formaldehyde (FA) not only inhibits the proliferative activity, but also causes DNA-protein crosslinks (DPCs) formation in bone marrow mesenchymal stem cells (BMSCs). The purpose of this study was to detect the protective effect of astragalus polysaccharide (APS) against the cytotoxicity and genotoxicity of BMSCs exposed to FA, and to explore potential molecular mechanisms of APS activity. Human BMSCs were cultured in vitro and randomly divided into control cells (Ctrl group), FA-treated cells (FA group, 120 μmol/L), and cells incubated with FA and increasing concentrations (40, 100, or 400 μg/mL) of APS (FA + APS groups). Cytotoxicity was measured by MTT assay. DNA strand breakage, DNA-protein crosslinks (DPCs), and micronucleus formation were respectively detected by comet assay, KCl-SDS precipitation assay, and micronucleus assay. The mRNA and protein expression level of xeroderma pigmentosum group A (XPA), xeroderma pigmentosum group C (XPC), excision repair cross-complementation group 1 (ERCC1), replication protein A1 (RPA1), and replication protein A2 (RPA2) were all detected by qRT-PCR and Western Blot. Compared with the FA group, the cytotoxicity, DNA strand breakage, DPCs, and micronucleus levels were decreased significantly in FA + APS groups (P < 0.01). Meanwhile, the mRNA and protein expression of XPA, XPC, ERCC1, RPA1, and RPA2 were up regulated significantly in the FA + APS groups (P < 0.05) with the most prominent effect of the 100 μg/mL APS. Our results suggest that APS can protect the cytotoxicity and genotoxicity of human BMSCs induced by FA. The mechanism may be associated with up-regulated expression of XPA, XPC, ERCC1, RPA1, and RPA2 in the nucleotide excision repair (NER) pathway which promotes DNA damage repair.

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3′-phospho-α,β-unsaturated aldehyde (PUA) and 5′-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3′-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5′-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5′-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.

Nucleosomal embedding reshapes the dynamics of abasic sites

Apurinic/apyrimidinic (AP) sites are the most common DNA lesions, which benefit from a most efficient repair by the base excision pathway. The impact of losing a nucleobase on the conformation and dynamics of B-DNA is well characterized. Yet AP sites seem to present an entirely different chemistry in nucleosomal DNA, with lifetimes reduced up to 100-fold, and the much increased formation of covalent DNA-protein cross-links leading to strand breaks, refractory to repair. We report microsecond range, all-atom molecular dynamics simulations that capture the conformational dynamics of AP sites and their tetrahydrofuran analogs at two symmetrical positions within a nucleosome core particle, starting from a recent crystal structure. Different behaviours between the deoxyribo-based and tetrahydrofuran-type abasic sites are evidenced. The two solvent-exposed lesion sites present contrasted extrahelicities, revealing the crucial role of the position of a defect around the histone core. Our all-atom simulations also identify and quantify the frequency of several spontaneous, non-covalent interactions between AP and positively-charged residues from the histones H2A and H2B tails that prefigure DNA-protein cross-links. Such an in silico mapping of DNA-protein cross-links gives important insights for further experimental studies involving mutagenesis and truncation of histone tails to unravel mechanisms of DPCs formation.

Abstract 5206: Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells

Abstract Bifunctional alkylating agents, such as cisplatin and several nitrosoureas, have clinical utility as antineoplastic drugs largely due to their DNA alkylating and crosslinking activity. Other drug candidates such as laromustine (VNP-40101M) and dianhydrogalacticol (VAL-083) have similar DNA-alkylating activities and have shown success in clinical trials against hard-to-treat cancers such as acute myelogenous leukemia and glioblastoma multiforme. While these agents' interstrand and instrastrand DNA crosslinking activities are widely cited as the critical cytotoxic event, their capacity to crosslink DNA to chromosomally-affiliated proteins may also contribute to their anticancer profile. Indeed, DNA-protein crosslinks (DPCs) are highly toxic phenomena that are difficult for cells to repair and severely compromise gene transcription and DNA replication. Herein, we assess concentration-dependent DPC formation by a wide variety of bifunctional alkylating agents in HL-60 human leukemia cells using two parallel approaches. In one approach, protein-bound DNA was quantified using PicoGreen after precipitating proteins from lysates of drug-treated cells and compared to total DNA concentration. In another, chromosomal DNA was extracted from lysates of drug-treated cells then treated with fluorescein isothiocyanate, which modifies primary amines on proteins. Protein concentration was measured by fluorescence and compared to that of the extracted DNA. The nitrogen mustard mechlorethamine and the chloroethyl nitrosoureas carmustine and lomustine were the most potent agents, yielding significant DPC formation at clinically relevant doses (10−5 M). Cisplatin and laromustine yielded DPCs at similar concentrations, but to a lesser extent. Dianyhydrogalacticol, epichlorohydrin, and diepoxybutane, which all rely on epoxide functional groups for attachment to cellular nucleophiles, required at least an order of magnitude higher drug concentrations for significant DPC observation. These data suggest bifunctional alkylating agents featuring halide leaving groups are most apt to crosslink DNA to nuclear proteins and may offer insights into patterns of cytotoxicity and clinical utility for this important class of chemotherapeuric agents. Citation Format: Hailey L. Cerrato, Sam W. Marchant, Allie H. Naccara, Thomas J. LaJoie, Shawnya Michaels, Dennis Brown, Kevin P. Rice. Quantifying DNA-protein crosslinks induced by bifunctional alkylating agents in cultured human leukemia cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5206.

DNA–protein crosslinks from environmental exposure: Mechanisms of formation and repair

Many environmental carcinogens cause DNA damage, which can result in mutations and other alterations in genomic DNA if not repaired promptly. Because of the bulkiness of the lesions, DNA-protein crosslinks (DPCs) are one of the types of toxic DNA damage with potentially deleterious consequences. Despite the importance of DPCs, how cells remove these complex DNA adducts has been incompletely understood. However, major progress in the DPC repair field over the past 5 years now supports the view that cells are equipped with multiple mechanisms to cope with DPCs. Here, we first provide an overview of environmental substances that induce DPCs, describing the sources of exposure and mechanisms of DPC formation. We then review current models of DPC repair and discuss their significance for environmental carcinogens.