Synthesis of Sequence-Specific DNA–Protein Conjugates via a Reductive Amination Strategy

DNA–protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to <i>bis</i>-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their superbulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty in generating site-specific DNA substrates representative of DPC lesions formed <i>in vivo</i>. In the present study, a novel approach involving postsynthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides

Major Groove Orientation of the (2<i>S</i>)‑<i>N</i>⁶‑(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the <i>N</i>⁶ exocyclic nitrogen of adenine to form <i>N</i>⁶-(hydroxy-3-buten-1-yl)-2′-dA ((2<i>S</i>)-<i>N</i>⁶-HB-dA) adducts (Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg, J. A. (1997) Carcinogenesis 18, 137−147). The structure of the (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct has been determined in the 5′-d­(C¹G²G³A⁴<u>C</u>^<u>5</u><u>Y</u>^<u>6</u><u>A</u>^<u>7</u>G⁸A⁹A¹⁰G¹¹)-3′:5′-d­(C¹²T¹³T¹⁴C¹⁵T¹⁶T¹⁷G¹⁸T¹⁹ C²⁰C²¹G²²)-3′ duplex [Y = (2<i>S</i>)-<i>N</i>⁶-HB-dA] containing codon 61 (underlined) of the human N-<i>ras</i> protooncogene, from NMR spectroscopy. The (2<i>S</i>)-<i>N</i>⁶-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the C_α carbon, the methylene protons of the modified nucleobase Y⁶ faced the 5′ direction, which placed the C_β carbon in the 3′ direction. The C_β hydroxyl group faced toward the solvent, as did carbons C_γ and C_δ. The C_β hydroxyl group did not form hydrogen bonds with either T¹⁶ <i>O</i>⁴ or T¹⁷ <i>O</i>⁴. The (2<i>S</i>)-<i>N</i>⁶-HB-dA nucleoside maintained the <i>anti</i> conformation about the glycosyl bond, and the modified base retained Watson–Crick base pairing with the complementary base (T¹⁷). The adduct perturbed stacking interactions at base pairs C⁵:G¹⁸, Y⁶:T¹⁷, and A⁷:T¹⁶ such that the Y⁶ base did not stack with its 5′ neighbor C⁵, but it did with its 3′ neighbor A⁷. The complementary thymine T¹⁷ stacked well with both 5′ and 3′ neighbors T¹⁶ and G¹⁸. The presence of the (2<i>S</i>)-<i>N</i>⁶-HB-dA resulted in a 5 °C reduction in the <i>T</i>_m of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove

Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

DNA–protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA–protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to <i>in vitro</i> primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts

Polymerase Bypass of <i>N</i>⁶‑Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene

<i>N</i>⁶-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine (<i>N</i>⁶-HB-dA I) and <i>N</i>⁶,<i>N</i>⁶-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (<i>N</i>⁶,<i>N</i>⁶-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the <i>N</i>⁶ position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the <i>N</i>⁶-H atom of adenine is required for Watson–Crick hydrogen bonding with thymine, <i>N</i>⁶-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived <i>N</i>⁶-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (<i>S</i>)-<i>N</i>⁶-HB-dA I and (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts were subjected to <i>in vitro</i> translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (<i>S</i>)-<i>N</i>⁶-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (<i>S</i>)-<i>N</i>⁶-HB-dA I. In contrast, hPol β was completely blocked by (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (<i>S</i>)-<i>N</i>⁶-HB-dA I was mostly error-free, replication of DNA containing (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (<i>S</i>)-<i>N</i>⁶-HB-dA I lesions are not miscoding, but that exocyclic (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson–Crick pairs with dT

Base Excision Repair of <i>N</i>⁶<i>-</i>Deoxyadenosine Adducts of 1,3-Butadiene

The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including <i>N</i>⁶-(2-hydroxy-3-buten-1-yl)-2′-deoxyadenosine (<i>N</i>⁶-HB-dA), 1,<i>N</i>⁶-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,<i>N</i>⁶-HMHP-dA), and <i>N</i>⁶,<i>N</i>⁶-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (<i>N</i>⁶,<i>N</i>⁶-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2′-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (<i>S</i>)-<i>N</i>⁶-HB-dA, (<i>R</i>,<i>S</i>)-1,<i>N</i>⁶-HMHP-dA, and (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (<i>S</i>)-<i>N</i>⁶-HB-dA > (<i>R</i>,<i>R</i>)-<i>N</i>⁶,<i>N</i>⁶-DHB-dA > (<i>R</i>,<i>S</i>)-1,<i>N</i>⁶-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells

DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA–protein cross-links (DPCs). Interstrand DNA–DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human <i>O</i>⁶-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT–DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles