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5-Formylcytosine (5fC) is an endogenous epigenetic DNA mark introduced via enzymatic oxidation of 5-methyl-dC in DNA. We and others recently reported that 5fC can form reversible DNA-protein conjugates with histone proteins, likely contributing to regulation of nucleosomal organization and gene expression. The protein component of DNA-protein cross-links can be proteolytically degraded, resulting in smaller DNA-peptide cross-links. Unlike full-size DNA-protein cross-links that completely block replication and transcription, DNA-peptide cross-links can be bypassed by DNA and RNA polymerases and can potentially be repaired via the nucleotide excision repair (NER) pathway. In the present work, we constructed plasmid molecules containing reductively stabilized, site-specific 5fC-polypeptide lesions and employed a quantitative MS-based assay to assess their effects on transcription in cells. Our results revealed that the presence of DNA-peptide cross-link significantly inhibits transcription in human HEK293T cells but does not induce transcription errors. Furthermore, transcription efficiency was similar in WT and NER-deficient human cell lines, suggesting that the 5fC-polypeptide lesion is a weak substrate for NER. This finding was confirmed by in vitro NER assays in cell-free extracts from human HeLa cells, suggesting that another mechanism is required for 5fC-polypeptide lesion removal. In summary, our findings indicate that 5fC-mediated DNA-peptide cross-links dramatically reduce transcription efficiency, are poor NER substrates, and do not cause transcription errors., (© 2019 Ji et al.)

The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.

One of the major biomarkers of oxidative stress and oxidative damage of cellular DNA is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is more easily oxidized than guanine to diverse oxidative products. In this work, we have investigated further oxidative transformations of 8-oxoGua in single- and double-stranded oligonucleotides to the dehydroguanidinohydantoin, oxaluric acid, and diastereomeric spiroiminodihydantoin lesions. The relative distributions of these end products were explored by a combined kinetic laser spectroscopy and mass spectrometry approach and are shown to depend markedly on the presence of superoxide radical anions. The 8-oxaGua radicals were produced by one-electron oxidation of 8-oxoGua by 2-aminopurine radicals generated by the two-photon ionization of 2-aminopurine residues site specifically positioned in 5'-d(CC[2-aminopurine]TC[8-oxoGua]CTACC). The hydrated electrons also formed in the photoionization process were trapped by dissolved molecular oxygen thus producing superoxide. A combination reaction between the 8-oxoGua and superoxide radicals occurs with the rate constant of (1.3 +/- 0.2) x 10(8) m(-1) s(-1) and (1.0 +/- 0.5) x 10(8) m(-1) s(-1) in single- and double-stranded DNA, respectively. The major end products of this reaction are the dehydroguanidinohydantoin lesions that slowly hydrolyze to oxaluric acid residues. In the presence of Cu,Zn-superoxide dismutase, an enzyme that induces the rapid catalytic dismutation of superoxide to the less reactive H(2)O(2) and O(2), the yields of the dehydroguanidinohydantion lesions become negligible. Under these conditions, the 8-oxoGua radicals do not exhibit any observable reactivities with oxygen (k < 10(2) m(-1) s(-1)), decay on the time interval of several seconds, and the major reaction products are the spiroiminodihydantoin lesions. The possible biological implications of the 8-oxoGua oxidation are discussed.

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol kappa. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N(2)-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.

DNA polymerase mu (Polmu) is a newly discovered member of the polymerase X family with unknown cellular function. The understanding of Polmu function should be facilitated by an understanding of its biochemical activities. By using purified human Polmu for biochemical analyses, we discovered the lesion bypass activities of this polymerase in response to several types of DNA damage. When it encountered a template 8-oxoguanine, abasic site, or 1,N(6)-ethenoadenine, purified human Polmu efficiently bypassed the lesion. Even bulky DNA adducts such as N-2-acetylaminofluorene-adducted guanine, (+)- and (-)-trans-anti-benzo[a]pyrene-N(2)-dG were unable to block the polymerase activity of human Polmu. Bypass of these simple base damage and bulky adducts was predominantly achieved by human Polmu through a deletion mechanism. The Polmu specificity of nucleotide incorporation indicates that the deletion resulted from primer realignment before translesion synthesis. Purified human Polmu also effectively bypassed a template cis-syn TT dimer. However, this bypass was achieved in a mainly error-free manner with AA incorporation opposite the TT dimer. These results provide new insights into the biochemistry of human Polmu and show that efficient translesion synthesis activity is not strictly confined to the Y family polymerases.

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.

The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair. To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo[a]pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed. DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct. Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision. Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo[a]pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision. In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM). Formation of this complex was absolutely dependent upon UvrC. Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction. The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate. Incision of this Y structure occurs even without DNA damage. Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease.