RESUMO
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.
Assuntos
Alquil e Aril Transferases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Peptídeos/química , HumanosRESUMO
Etheno (ε)-adducts, e.g., 1,N2-ε-guanine (1,N2-ε-G) and 1,N6-ε-adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N2-ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N2-ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with nonhydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5'-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N2-ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N2-ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication.
Assuntos
Adutos de DNA/química , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiguanosina/análogos & derivados , Cromatografia Líquida , Cristalografia por Raios X , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Humanos , Espectrometria de Massas em TandemRESUMO
Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, â³, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol â³. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol â³ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/farmacologia , Cromatografia Líquida/métodos , DNA/química , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Dibrometo de Etileno/química , Humanos , Cinética , Estrutura Molecular , Mutação , Nucleotídeos/genética , Peptídeos/genética , Espectrometria de Massas em Tandem/métodosRESUMO
In the course of studies on the enhancement of 1,2-dibromoethane-induced DNA base pair mutations by O6-alkylguanine-DNA alkyltransferase (AGT, MGMT), we discovered the facile reaction of AGT with an abasic site in DNA, leading to covalent cross-linking. The binding of AGT differs from the mechanism reported for the protein HMCES; instead it appears to involve formation of a stable thioglycoside. Facile cross-linking was also observed with the protease papain, which like AGT has a low pKa cysteine, and the tripeptide glutathione.
Assuntos
Dibrometo de Etileno , Tioglicosídeos , Cisteína , DNA/metabolismo , Reparo do DNA , Glutationa , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Papaína/metabolismo , Compostos de SulfidrilaRESUMO
Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2-mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.
Assuntos
Adenosina/análogos & derivados , Dano ao DNA , Reparo do DNA , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ribonuclease H/metabolismoRESUMO
Acanthamoeba polyphaga mimivirus is an amoeba-infecting giant virus with over 1000 genes including several involved in DNA replication and repair. Here, we report the biochemical characterization of gene product 577 (gp577), a hypothetical protein (product of L537 gene) encoded by mimivirus. Sequence analysis and phylogeny suggested gp577 to be a primase-polymerase (PrimPol)-the first PrimPol to be identified in a nucleocytoplasmic large DNA virus (NCLDV). Recombinant gp577 protein purified as a homodimer and exhibited de novo RNA as well as DNA synthesis on circular and linear single-stranded DNA templates. Further, gp577 extends a DNA/RNA primer annealed to a DNA or RNA template using deoxyribonucleoties (dNTPs) or ribonucleotides (NTPs) demonstrating its DNA/RNA polymerase and reverse transcriptase activity. We also show that gp577 possesses terminal transferase activity and is capable of extending ssDNA and dsDNA with NTPs and dNTPs. Mutation of the conserved primase motif residues of gp577 resulted in the loss of primase, polymerase, reverse transcriptase and terminal transferase activities. Additionally, we show that gp577 possesses translesion synthesis (TLS) activity. Mimiviral gp577 represents the first protein from an NCLDV endowed with primase, polymerase, reverse transcriptase, terminal transferase and TLS activities.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mimiviridae/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/genética , DNA Nucleotidilexotransferase/isolamento & purificação , DNA Primase/química , DNA Primase/genética , DNA Primase/isolamento & purificação , Primers do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Dimerização , Mimiviridae/genética , RNA , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Linhagem Celular , Cristalografia por Raios X , Primers do DNA/química , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
We report the synthesis of N2-aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N2-dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N2-aryl-dG damaged DNAs by Pol IV.
Assuntos
DNA Polimerase beta/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/síntese química , Escherichia coli/enzimologia , Dano ao DNA , DNA Polimerase beta/química , Replicação do DNA , Desoxiguanosina/química , Escherichia coli/químicaRESUMO
DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N2-2'-deoxyguanosine (N2-dG) and N6-2'-deoxyadenosine (N6-dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N2-dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.
Assuntos
Antraquinonas/metabolismo , Carcinógenos/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Antraquinonas/química , Carcinógenos/química , Cristalografia por Raios X , Adutos de DNA/química , Adutos de DNA/genética , DNA Polimerase Dirigida por DNA/química , Humanos , Modelos MolecularesRESUMO
N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by â¼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.
Assuntos
Adutos de DNA/química , DNA de Forma B/química , DNA/química , Desoxiguanosina/análogos & derivados , Escherichia coli/química , Pareamento de Bases , DNA/metabolismo , Adutos de DNA/metabolismo , DNA de Forma B/metabolismo , Desoxiguanosina/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica MolecularAssuntos
Toxicologia/história , Dano ao DNA , História do Século XX , História do Século XXI , Humanos , Estados UnidosRESUMO
Lucidin is a genotoxic and mutagenic hydroxyanthraquinone metabolite, which originates from the roots of Rubia tinctorum L. (madder root). It reacts with exocyclic amino groups of DNA nucleobases and forms adducts/lesions leading to carcinogenesis. To study the effect of lucidin-induced DNA damage, herein, we report the first synthesis of a structural analogue of lucidin [N(2)-methyl-(1,3-dimethoxyanthraquinone)-deoxyguanosine, LdG] embedded DNAs utilizing phosphoramidite strategy. LdG modification in a DNA duplex imparts destabilization (ΔTm â¼5 °C/modification), which is attributed to the unfavorable contribution from the enthalpy. Primer extension studies using the Klenow fragment (exo(-)) of Escherichia coli DNA polymerase I demonstrate that bypass of LdG modification is error prone as well as slow compared to that across the unmodified sites. Molecular dynamics simulations of the binary complex of Bacillus fragment polymerase (homologue of the Klenow fragment) and LdG-DNA duplex elucidate the structural fluctuations imparted by the LdG lesion, as well as the molecular mechanism of bypass at the lesion site. Overall, the results presented here show that the lucidin adduct destabilizes DNA structure and reduces fidelity and processivity of DNA synthesis.
Assuntos
Antraquinonas/farmacologia , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiguanosina/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , Desoxiguanosina/química , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven N2-dG lesions of different steric bulkiness. In the presence of Mg2+ ions, bulky N2-isobutyl-dG, N2-benzyl-dG, N2-methyl(1-naphthyl)-dG, N2-methyl(9-anthracenyl)-dG, N2-methyl(1-pyrenyl)-dG, and N2-methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite N2-ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn2+ ions. When Mn2+ replaced Mg2+, PrimPol carried out DNA synthesis on all DNA templates with N2-dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite N2-ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the N2-isobutyl-dG and N2-benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.
Assuntos
Dano ao DNA , Desoxicitidina Monofosfato , Humanos , Desoxiguanosina/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Adutos de DNA , Proteínas Nucleares/metabolismo , DNA Primase/metabolismo , Enzimas Multifuncionais/metabolismoRESUMO
Etheno (and ethano) derivatives of nucleic acid bases have an extra 5-membered ring attached. These were first noted as wyosine bases in tRNAs. Some were fluorescent, and the development of etheno derivatives of adenosine, cytosine, and guanosine led to the synthesis of fluorescent analogs of ATP, NAD+, and other cofactors for use in biochemical studies. Early studies with the carcinogen vinyl chloride revealed that these modified bases were being formed in DNA and RNA and might be responsible for mutations and cancer. The etheno bases are also derived from other carcinogenic vinyl monomers. Further work showed that endogenous etheno DNA adducts were present in animals and humans and are derived from lipid peroxidation. The chemical mechanisms of etheno adduct formation involve reactions with bis-electrophiles generated by cytochrome P450 enzymes or lipid peroxidation, which have been established in isotopic labeling studies. The mechanisms by which etheno DNA adducts miscode have been studied with several DNA polymerases, aided by the X-ray crystal structures of these polymerases in mispairing situations and in extension beyond mispairs. Repair of etheno DNA adduct damage is done primarily by glycosylases and also by the direct action of dioxygenases. Some human DNA polymerases (η, κ) can insert bases opposite etheno adducts in DNA and RNA, and the reverse transcriptase activity may be of relevance with the RNA etheno adducts. Further questions involve the extent that the etheno adducts contribute to human cancer.
RESUMO
The N2 -position of 2'-deoxyguanosine (N2 -position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N2 -dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N2 -dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N2 -dG damaged DNAs. This article describes a detailed protocol of the synthesis of N2 -aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N2 -dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N2 -dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N2 -position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc.
Assuntos
Desoxiguanosina/química , Oligonucleotídeos/síntese química , Compostos Organofosforados/síntese química , Dano ao DNA , Instabilidade Genômica , Oligonucleotídeos/química , Compostos Organofosforados/químicaRESUMO
O6 -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.