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![Formation of an hAGT-DBE adduct. In A, hAGT protein (squares) and C145S mutant (circles) (1– 40 g) were incubated with 20 mM of [ 14 C]DBE (10 Ci/mol) in the presence (open symbols) or absence (filled symbols) of ctDNA for 30 min. The amounts of [ 14 C] transferred to the protein were examined, and the amount of DBE bound to the AGT added was calculated. The AGT concentration was calculated using a molecular weight value for the recombinant hAGT of 21,862. In B, to estimate the rate of adduct formation, 20 g of hAGT was incubated with 0 –25 mM [ 14 C]DBE for 0 –30 min. The rate of reaction was determined from the amount of [ 14 C] bound to the protein and then plotted against the DBE concentration.](publication/11232183/figure/fig4/AS:460279013220355@1486750476431/Formation-of-an-hAGT-DBE-adduct-In-A-hAGT-protein-squares-and-C145S-mutant-circles.png)
Formation of an hAGT-DBE adduct. In A, hAGT protein (squares) and C145S mutant (circles) (1– 40 g) were incubated with 20 mM of [ 14 C]DBE (10 Ci/mol) in the presence (open symbols) or absence (filled symbols) of ctDNA for 30 min. The amounts of [ 14 C] transferred to the protein were examined, and the amount of DBE bound to the AGT added was calculated. The AGT concentration was calculated using a molecular weight value for the recombinant hAGT of 21,862. In B, to estimate the rate of adduct formation, 20 g of hAGT was incubated with 0 –25 mM [ 14 C]DBE for 0 –30 min. The rate of reaction was determined from the amount of [ 14 C] bound to the protein and then plotted against the DBE concentration.
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![FIG. 3. Repair of S-[2-(O 6 -ethylguanyl)]glutathione by hAGT in vitro....](publication/11232183/figure/fig3/AS:460279013220354@1486750476402/Repair-of-S-2-O-6-ethylguanylglutathione-by-hAGT-in-vitro-A-5-end-32-P-labeled_Q320.jpg)
The presence of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) paradoxically increases the mutagenicity and cytotoxicity of 1,2-dibromoethane (DBE) in Escherichia coli. This enhancement of genotoxicity did not occur when the inactive C145A mutant of human AGT (hAGT) was used. Also, hAGT did not enhance the genotoxicity of S-(2-...
Citations
... 35−37 With low M r thiols, the questions of polymerase retardation and miscoding remain to be addressed. Other questions are how a DNA polymerase can bypass a very large entity, i.e., a DNA-protein cross-link, 13,14,[19][20][21]38 as well as what determines the binding to the AP sites: thiol pK a , nucleophilicity, DNA affinity, concentration, or a combination of all of these. Further studies are in progress. ...
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.
... The DNA repair protein O 6alkylguanine DNA-alkyltransferase (AGT or MGMT) is known to form cross-links with DNA in the presence of bifunctional electrophiles (i.e., 1,2-dibromoethane) resulting in G:C to T:A transversions and other mutations in both Escherichia coli and CHO cells (34)(35)(36). The mechanism of formation of AGT-DNA cross-links involves the nucleophilic attack of the active site residue Cys-145 on 1,2-dibromoethane and leads to the formation of a half-mustard intermediate, which further cyclizes into an unstable episulfonium ion (37). ...
... The mechanism of formation of AGT-DNA cross-links involves the nucleophilic attack of the active site residue Cys-145 on 1,2-dibromoethane and leads to the formation of a half-mustard intermediate, which further cyclizes into an unstable episulfonium ion (37). Nucleophilic sites on DNA react with the unstable episulfonium ion and form AGT-DNA cross-links (34,35), including the N6 position of dA, N7 position of dG, N2 position of dG, N1 position of dG, and O6 position of dG (37). This process is related to the induction of mutations in both E. coli and mammalian cells (34)(35)(36). ...
... Nucleophilic sites on DNA react with the unstable episulfonium ion and form AGT-DNA cross-links (34,35), including the N6 position of dA, N7 position of dG, N2 position of dG, N1 position of dG, and O6 position of dG (37). This process is related to the induction of mutations in both E. coli and mammalian cells (34)(35)(36). ...
DNA-protein crosslinks 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 crosslinks 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, we found that 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 crosslinked 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.
... Ethylene dibromide (EDB, or 1,2dibromoethane) is a known carcinogen that had formerly been used as a gasoline additive, soil fumigant, and pesticide (44)(45)(46)(47). Overexpression of AGT paradoxically increased EDB toxicity and the incidence of base pair mutations in bacterial and mammalian cells (48,49). Cys-145 of AGT reacts with EDB via a nucleophilic substitution that results in a halfmustard intermediate, which cyclizes to form an unstable episulfonium ion that can form a covalent bond with DNA ( Fig. 1). ...
Unrepaired DNA-protein crosslinks, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein crosslinks can be cleaved into DNA-peptide crosslinks, 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 crosslink 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, crosslinked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA crosslink 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, 10-fold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the crosslinked 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.
... DPCs are common DNA lesions generated by various endogenous and exogenous agents, including aldehyde metabolites, ionizing radiation and UV light, formaldehyde, 1,3-butadiene, 1,2-dibromoethane, transition metals, and bifunctional chemotherapeutic drugs such as nitrogen mustards and platinum compounds. Various proteins including O 6 -alkylguanine DNA alkyltransferase (AGT), topoisomerase I, topoisomerase II, and DNA polymerase β have been shown to form DPCs. (183) Because DPCs are bulky lesions, it can interfere with normal physiological processes including replication, transcription, DNA repair, and chromatin remodeling. Genetic and biochemical studies have shown that both nucleotide excision repair (NER) and homologous recombination (HR) could remove DPCs and both pathways play different roles. ...
Nucleic acids are prone to fragmentation in the ionization process of mass spectrometry (MS). The cause can be found in the polar nature of this analyte class. Following the introduction of the so‐called soft‐ionization techniques of electrospray ionization (ESI) and matrix‐assisted laser desorption ionization (MALDI) at the end of the 1980s, mass spectrometric analysis of nucleic acids has gained broader applicability. In combination with further developments of the associated instrumentation and optimized preparation and purification methods, a dramatic enhancement of the mass range, detection sensitivity, and resolution became possible in routine analysis of oligonucleotides, up to a length of 50 nucleotides. At present, the necessary quantity of sample is in the subfemtomole range.
MS is an accurate, rapid, and sensitive tool ideally suited for sequencing shorter nucleic acids. MS sequencing holds great promise in clinical diagnostics, forensics, paternity analysis, livestock breeding, plant cultivation, and cell line typing. In addition, MS has been used for various other important applications including analysis of noncovalent complexes, mixture analysis, different sequencing strategies, misincorporation analysis of translesion DNA synthesis products, detection and mapping of DNA lesions, detection and characterization of DNA‐protein crosslinks (DPCs), and clinical diagnostics. The use of MS for the detection of DNA adducts in oligonucleotides is extremely useful because it has not only eliminated the need for hydrolysis but also allowed to map the position of the adduct and determine the sequence of the oligonucleotide. This article describes the fundamentals of mass spectrometric analyses of nucleic acids and some of its common applications. The primary focus is on LC‐ESI‐tandem MS and MALDI/MS.
... Interestingly, the cytotoxic and mutagenic effects of 1,2-dibromoethane, butadiene diepoxide, and epibromohydrin in Escherichia coli and Chinese hamster cells are significantly increased by the ectopic overexpression of human O 6 -alkylguanine-DNA alkyltransferase (hAGT), the primary function of which is to maintain genomic integrity by directly reversing alkylation DNA damage [48,49]. These agents cross-link hAGT and DNA to form type 1 DPCs together with other DNA damage [50,51]. An initial reaction occurs between the hAGT and one side of the reagents to produce a reactive intermediate at Cys145 at the active site of hAGT. ...
... An initial reaction occurs between the hAGT and one side of the reagents to produce a reactive intermediate at Cys145 at the active site of hAGT. The resulting intermediate subsequently attacks the N7 of guanine in DNA, yielding covalent hAGT-DNA cross-links [50,51]. It has been proposed that the hAGT-DNA cross-links and/or apurinic/apyrimidinic (AP) sites arising from the depurination of hAGT-DNA cross-links are involved in the cytotoxicity and mutagenicity observed in the presence of hAGT. ...
... DNA-protein crosslinks (DPCs) are common DNA lesions generated by various endogenous and exogenous agents and proteins. Proteins including AGT, topoisomerase I, topoisomerase II, and DNA polymerase β have been shown to form DPCs (Liu et al., 2002). DPCs are bulky lesions that can impart significant toxicity by blocking replication and transcription or even causing mutations. ...
... DNA-protein crosslinks (DPCs) are common DNA lesions generated by various endogenous and exogenous agents, including aldehyde metabolites, physical damage such as ionizing radiation and UV light, chemical agents including formaldehyde, 1,3-butadiene, 1,2dibromoethane, transition metals, and bifunctional chemotherapeutic drugs such as nitrogen mustards and platinum compounds. Proteins including AGT, topoisomerase I, topoisomerase II, and DNA polymerase β have been shown to form DPCs (Liu et al., 2002). DPCs are bulky lesions that can disrupt normal physiological processes including replication, transcription, DNA repair, and chromatin remodeling. ...
This unit contains a complete procedure for the detection and structural characterization of DNA protein crosslinks (DPCs). The procedure also describes an approach for the quantitation of the various structurally distinct DPCs. Although various methods have been described in the literature for labile DPCs, characterization of nonlabile adducts remain a challenge. Here we present a novel approach for characterization of both labile and non-labile adducts by the use of a combination of chemical, enzymatic, and mass spectrometric approaches. A Raney Ni-catalyzed reductive desulfurization method was used for removal of the bulky peptide adducts, enzymatic digestion was used to digest the protein to smaller peptides and DNA to nucleosides, and finally LC-ESI-tandem mass spectrometry (MS) was utilized for detection and characterization of nucleoside adducts. © 2015 by John Wiley & Sons, Inc.
Copyright © 2015 John Wiley & Sons, Inc.
... Multiple studies have shown that AGT readily participates in crosslinking reactions with DNA in the presence of bis-electrophiles to produce toxic and mutagenic DPC lesions. [11][12][13][14] AGT overexpression in bacteria enhances the toxicity and mutagenicity of dihaloalkanes such as 1,2-dibromoethane (DBE), [11,[15][16][17][18] and the introduction of DNAreactive AGT monoepoxides into mammalian cells via electroporation leads to mutations and cell death. [19] It has been proposed that AGT Cys 145 reacts with dihaloalkanes to generate a half mustard, which alkylates DNA via an episulfonium ion intermediate to generate covalent DPCs. ...
... [19] It has been proposed that AGT Cys 145 reacts with dihaloalkanes to generate a half mustard, which alkylates DNA via an episulfonium ion intermediate to generate covalent DPCs. [10,18,20,21] The majority of the resulting lesions (> 80%) are hydrolytically labile adducts involving the N7 position of guanine in DNA, however, hydrolytically stable DPC lesions are also formed. Gel shift studies have revealed that DBE forms covalent cross-links between AGT and all four DNA bases, with the adduct yields in the order G > T > C > A. [20] However, the structures of hydrolytically stable AGT-DNA adducts have remained elusive. ...
... [13,14] Subsequent proteolytic digestion of the protein generates nucleobase-peptide conjugates, which can be readily sequenced by tandem mass spectrometry to identify the cross-linking site. [13,14,18,20] This selective hydrolysis approach has been used to examine AGT cross-linking to the N7 position of guanine in DNA in the presence of 1,2,3,4-diepoxybutane, epihalohydrins, dihaloalkanes, nitrogen mustards, and platinum compounds. [13,14,21,23] However, hydrolytically stable DPC adducts may be equally important for the toxicity and mutagenicity of cross-linking agents. ...
Easier with ethyl: Guengerich and co-workers have developed a powerful new approach to the structure elucidation of hydrolytically stable AGT-DNA crosslinks by reductive desulfurization of the thioether linkage between AGT and DNA to convert cysteine DPCs to the corresponding ethyl-DNA adducts, which can be readily characterized by LC-MS(n) .
... [1,2] Although AGT is a repair protein, it has been shown to paradoxically augment the toxicity of 1,2-dibromoethane (DBE, also called ethylene dibromide) (Scheme 1). [3] Overexpression of human AGT (hAGT) or AGTs from other species enhances the mutagenicity and lethality of DBE in Escherichia coli and mammalian cells. [3] This unusual enhancement of DBE toxicity stems from the increased reactivity of the active site Cys145 residue that readily reacts with the bis-electrophile DBE to form a half-mustard. ...
... [3] Overexpression of human AGT (hAGT) or AGTs from other species enhances the mutagenicity and lethality of DBE in Escherichia coli and mammalian cells. [3] This unusual enhancement of DBE toxicity stems from the increased reactivity of the active site Cys145 residue that readily reacts with the bis-electrophile DBE to form a half-mustard. The half-mustard intermediate subsequently cyclizes to yield an episulfonium ion that can react to form a covalent DNAethylene-AGT crosslink (based on the demonstrated chemistry with glutathione (GSH)) (Scheme 1). ...
A combination of chemical modifications and LC-tandem MS was used for the structure elucidation of various ethylene crosslinks of DNA with O(6) -alkylguanine-DNA alkyltransferase (AGT, see picture). The elucidation of the chemical structures of such DNA-protein crosslinks is necessary to understand mechanisms of mutagenesis.
... For example, it has been reported that the cytotoxicity and mutagenicity of several bifunctional alkylation agents including 1,2-dibromoethane, dibromomethane, and DEB, are enhanced in bacteria which over-express human O 6alkylguanine DNA alkyltransferase (AGT) protein due to the formation of toxic AGT-DNA cross-links. 15,16,15,17 AGT is a DNA repair protein that typically protects the human genome from the damaging effects of promutagenic O 6 -alkylguanine lesions induced by simple alkylating agents. 18 During the repair reaction, the O 6 -alkylguanine nucleotide is flipped out of the base stack into the protein's active site, where it is subject to nucleophilic attack by the activated side chain thiolate anion of Cys 145. ...
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 O6-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. Lower 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.
... Table S2 lists the mutation types, mutation sites, base changes and amino acid changes of the mutation spectra and Table S3 lists the mutation frequency, total analyzed samples, total mutants and mutation number of specific base pair. The mutation spectra in GWR109 cells (Table 1) are similar to those reported previously 20 . A previous study showed that the mutations at G:C sites are produced by hAGT crosslinked with G base of DNA 18 . ...
Bis-electrophiles including dibromoethane and epibromohydrin can react with O(6)-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA-protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.
