N2 -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase ?.
ABSTRACT: N2 -Alkyl-2'-deoxyguanosine triphosphate (N2 -alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N2 -Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase?? (pol??), but was a poor substrate for pols??, ?, ?, ?, or ?. In?vivo reactivity was evaluated through incubation of N2 -4-ethynylbenzyl-dG with wild-type and pol?? deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)-FAM-azide demonstrated that only cells containing pol?? were able to incorporate N2 -4-ethynylbenzyl-dG into the nucleus. This is the first instance of a Y-family-polymerase-specific dNTP, and this method could be used to probe the activity of pol?? in?vivo.
Project description:DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O6-dG-derived ICLs. Although less abundant, O6-alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O6-2'-deoxyguanosine-butylene-O6-2'-deoxyguanosine (O6-dG-C4-O6-dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O6-dG-C4-O6-dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol ? is completely inhibited by O6-dG-C4-O6-dG ICLs. Moderate bypass activities are observed for human pol ? and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol ? exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol ? in O6-dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol ?. Together, this study provides the first set of biochemical evidence for the mutagenicity of O6-dG-derived ICLs.
Project description:The mechanistic aspects of the methylation of guanine in DNA initiated by methyl radicals that are derived from the metabolic oxidation of some chemical carcinogens remain poorly understood. In this work, we investigated the kinetics and the formation of methylated guanine products by two methods: (i) the combination of *CH3 radicals and guanine neutral radicals, G(-H)*, and (ii) the direct addition of *CH3 radicals to guanine bases. The simultaneous generation of *CH3 and dG(-H)* radicals was triggered by the competitive one-electron oxidation of dimethyl sulfoxide (DMSO) and 2'-deoxyguanosine (dG) by photochemically generated sulfate radicals in deoxygenated aqueous buffer solutions (pH 7.5). The photolysis of methylcob(III)alamin to form *CH3 radicals was used to investigate the direct addition of these radicals to guanine bases. The major end products of the radical combination reactions are the 8-methyl-dG and N2-methyl-dG products formed in a ratio of 1:0.7. In contrast, the methylation of dG by *CH3 radicals generates mostly the 8-methyl-dG adduct and only minor quantities of N2-methyl-dG (1:0.13 ratio). The methylation of the self-complementary 5'-d(AACGCGAATTCGCGTT) duplexes was achieved by the selective oxidation of the guanines with carbonate radical anions in the presence of DMSO as the precursor of *CH3 radicals. The methyl-G lesions formed were excised by the enzymatic digestion and identified by LC-MS/MS methods using uniformly 15N-labeled 8-methyl-dG and N2-methyl-dG adducts as internal standards. The ratios of 8-methyl-G/N2-methyl-G lesions derived from the combination of methyl radicals with G(-H)* radicals positioned in double-stranded DNA or that with the free nucleoside dG(-H)* radicals were found to be similar. Utilizing the photochemical method and dipropyl or dibutyl sulfoxides as sources of alkyl radicals, the corresponding 8-alkyl-dG and N2-alkyl-dG adducts were also generated in ratios similar to those obtained with DMSO.
Project description:4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O6-methyl-2'-deoxyguanosine (O6-Me-dG), O6-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O6-POB-dG), O2-methylthymidine (O2-Me-dT), and O2-POB-dT. We evaluated the ability of human DNA polymerase ? to bypass this damage to evaluate the structural constraints on substrates for pol ? and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ? on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O6-Me-dG is a good substrate for pol ?, while O6-POB-dG and the O2-alkyl-dT adducts are poor substrates for pol ?. The reactivity with O6-Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ? toward O6-Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ? is unlikely to bypass O6-POB-dG or the O2-alkyl-dTs in vivo.
Project description: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) ? in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol ?, pol ?, pol ?, 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 ? efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol ?, pol ? 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 ? illustrate that the LdG adduct is accommodated by pol ? at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol ? 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.
Project description:Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ? or Pol ?. Genetic ablation of Pol ?, Pol ?, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G ? A transition and G ? T transversion mutations for these lesions. Moreover, further depletion of Pol ? in Pol ?- or Pol ?-deficient cells gives rise to elevated rates of G ? A and G ? T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.
Project description:Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²? as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (?200-1200 nM). DNA binding is enhanced 34-fold by Mn²? (K(d,DNA) = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn²? compared to Mg²? based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ?100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ?1.7-fold more accurate when Mg²? is the cofactor compared to Mn²?. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn²?) to 6-fold (Mg²?) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ?25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ?2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ?0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²?, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.
Project description:O 6-alkyl-2'-deoxyguanosine (O 6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector-based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O 6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines. We found that the extent of replication-blocking effects of these lesions is influenced by the size of the alkyl groups situated on the O 6 position of the guanine base. We also noted involvement of distinct DNA repair pathways and translesion synthesis polymerases (Pols) in ameliorating the replication blockage effects elicited by the straight- and branched-chain O 6-alkyl-dG lesions. We observed that O 6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O 6 position of guanine, whereas repair of the branched-chain lesions relied on nucleotide excision repair. Moreover, these lesions were highly mutagenic during cellular replication and exclusively directed G?A mutations; Pol ? and Pol ? participated in error-prone bypass of the straight-chain lesions, whereas Pol ? preferentially incorporated the correct dCMP opposite the branched-chain lesions. Together, these results uncover key cellular proteins involved in repair and translesion synthesis of O 6-alkyl-dG lesions and provide a better understanding of the roles of these types of lesions in the etiology of human cancer.
Project description:Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N2-position of guanine to produce N2-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2'-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2'-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2'-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N2-BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N2-BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.
Project description:Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol ?, pol ?, pol ?, pol ?, or Rev1. Up to 75% reduction in TLS occurred when pol ?, pol ?, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G ? T, as was reported for dG-C8-IQ. In each site, knockdown of pol ? resulted in an increase in MF, whereas MF was reduced when pol ?, pol ?, pol ?, or Rev1 was knocked down. The reduction in MF was most pronounced when pol ?, pol ?, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol ? predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols ?, pol ?, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ? and ? showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.
Project description:3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G?T>G?A>G?C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol ?, pol ?, pol ?, pol ?, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ?55% reduction) in the level of TLS occurred in cells with knockdown of pol ?. Pol ? and pol ? were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ? appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ? knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.