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1.  Sequence Context Modulation of Polycyclic Aromatic Hydrocarbon-Induced Mutagenesis 
DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N6-BPDE dA adducts at the second position in the N-ras codon 61(CAA) (612) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation, but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10S-BPDE and C10R-BPDE lesions linked through the N6 position of adenine in the sequence context N-ras codon 61, position 3 (C10S-BPDE and C10R-BPDE at 613) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10S-BPDE and C10R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis, but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 613 resulted in truncated products, with the C10S-BPDE adducts being more blocking than C10R-BPDE lesions, and termination of synthesis was more pronounced at position 613 than at 612 for each of the lesions.
doi:10.1002/em.21806
PMCID: PMC4118935  PMID: 23913516
BPDE; DNA polymerases; mutagenesis
2.  Mutagenic Spectra Arising from Replication Bypass of the 2,6-diamino-4-hydroxy-N5-methyl Formamidopyrimidine Adduct in Primate Cells 
Chemical research in toxicology  2013;26(7):1108-1114.
DNA exposures to electrophilic methylating agents that are commonly used during chemotherapeutic treatments cause diverse chemical modifications of nucleobases, with reaction at N7-dG being the most abundant. Although this base modification frequently results in destabilization of the glycosyl bond and spontaneous depurination, the adduct can react with hydroxide ion to yield a stable, ring-opened MeFapy-dG and this lesion has been reported to persist in animal tissues. Results from prior in vitro replication bypass investigations of the MeFapy-dG adduct had revealed complex spectra of replication errors that differed depending on the identity of DNA polymerase and the local sequence context. In this study, a series of nine site-specifically modified MeFapy-dG-containing oligodeoxynucleotides were engineered into a shuttle vector and subjected to replication in primate cells. In all nine sequence contexts examined, MeFapy-dG was shown to be associated with a strong mutator phenotype, predominantly causing base substitutions, with G to T transversions being most common. Single and dinucleotide deletions were also found in a subset of the sequence contexts. Interestingly, single-nucleotide deletions occurred not only at the adducted site, but also one nucleotide downstream of the adduct. Standard models for primer-template misalignment could account for some, but not all mutations observed. These data demonstrate that in addition to mutagenesis predicted from replication of DNAs containing O6-Me-dG and O4-Me-dT, the MeFapy-dG adduct likely contributes to mutagenic events following chemotherapeutic treatments.
doi:10.1021/tx4001495
PMCID: PMC3830661  PMID: 23763662
3.  Modulation of the Processive Abasic Site Lyase Activity of a Pyrimidine Dimer Glycosylase 
DNA repair  2011;10(10):1014-1022.
The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.
doi:10.1016/j.dnarep.2011.07.015
PMCID: PMC3185154  PMID: 21889915
Abasic sites; base excision repair; structure-function; protein engineering
4.  The leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity 
Chemical research in toxicology  2013;26(2):221-232.
Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments, and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ~16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC50 values, the exception being a six- to eight-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. Based on Michaelis-Menten kinetic analyses and DNA binding assays the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.
doi:10.1021/tx300392m
PMCID: PMC3578163  PMID: 23305233
DNA replication; polymerase; translesion DNA synthesis; small-molecule inhibitor
5.  Inhibition of DNA Glycosylases via Small Molecule Purine Analogs 
PLoS ONE  2013;8(12):e81667.
Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.
doi:10.1371/journal.pone.0081667
PMCID: PMC3857224  PMID: 24349107
6.  Inhibition of HIV-1 Reverse Transcriptase-Catalyzed Synthesis by Intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide Adducts 
PLoS ONE  2013;8(9):e72131.
To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10R and C10S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.
doi:10.1371/journal.pone.0072131
PMCID: PMC3778021  PMID: 24069141
7.  Ring-Opening of the γ-OH-PdG Adduct Promotes Error-Free Bypass by the Sulfolobus solfataricus DNA Polymerase Dpo4 
Chemical Research in Toxicology  2013;26(9):1348-1360.
Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N2-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus Dpo4, the prototypic Y-family DNA polymerase, using templates that contained the adduct in either the 5′-CXG-3′ or the 5′-TXG-3′ sequence context. Although γ-OH-PdG partially blocked Dpo4-catalyzed DNA synthesis, full primer extension was observed, and the majority of bypass products were error-free. Conversion of the adduct into an irreversibly ring-opened derivative prior to reaction facilitated bypass and further improved the fidelity. Structures of ternary Dpo4·DNA·dNTP complexes were determined with primers that either were positioned immediately upstream of the lesion (preinsertion complexes) or had a 3′-terminal dC opposite the lesion (postinsertion complexes); the incoming nucleotides, either dGTP or dATP, were complementary to the template 5′-neighbor nucleotide. In both postinsertion complexes, the adduct existed as ring-opened species, and the resulting base-pair featured Watson–Crick hydrogen bonding. The incoming nucleotide paired with the 5′-neighbor template, while the primer 3′-hydroxyl was positioned to facilitate extension. In contrast, γ-OH-PdG was in the ring-closed form in both preinsertion complexes, and the overall structure did not favor catalysis. These data provide insights into γ-OH-PdG chemistry during replication bypass by the Dpo4 DNA polymerase and may explain why γ-OH-PdG-induced mutations due to primer–template misalignment are uncommon.
doi:10.1021/tx400200b
PMCID: PMC3775444  PMID: 23947567
8.  Replication bypass of N2-deoxyguanosine interstrand cross-links by human DNA polymerases η and ι 
Chemical research in toxicology  2012;25(3):755-762.
DNA-interstrand cross-links (ICLs) can be repaired by biochemical pathways requiring DNA polymerases that are capable of translesion DNA synthesis (TLS). The anticipated function of TLS polymerases in these pathways is to insert nucleotides opposite and beyond the linkage site. The outcome of these reactions can be either error-free or mutagenic. TLS-dependent repair of ICLs formed between the exocyclic nitrogens of deoxyguanosines (N2-dG) can result in low-frequency base substitutions, predominantly G to T transversions. Previously, we demonstrated in vitro that error-free bypass of a model acrolein-mediated N2-dG ICL can be accomplished by human polymerase (pol) κ, while Rev1 can contribute to this bypass by inserting dC opposite the cross-linked dG. The current study characterized two additional human DNA polymerases, pol η and pol ι, with respect to their potential contributions to either error-free or mutagenic bypass of these lesions. In the presence of individual dNTPs, pol η could insert dA, dG, and dT opposite the cross-linked dG, but incorporation of dC was not apparent. Further primer extension was observed only from the dC and dG 3'; termini and the amounts of products were low relative to the matched undamaged substrate. Analyses of bypass products beyond the adducted site revealed that dG was present opposite the cross-linked dG in the majority of extended primers and short deletions were frequently detected. When pol ι was tested for its ability to replicate past this ICL, the correct dC was preferentially incorporated, but no further extension was observed. Under the steady-state conditions, the efficiency of dC incorporation was reduced ∼500-fold relative to the undamaged dG. Thus, in addition to pol κ-catalyzed error-free bypass of N2-dG ICLs, an alternative, albeit low-efficiency, mechanism may exist. In this pathway, either Rev1 or pol ι could insert dC opposite the lesion, while pol η could perform the subsequent extension.
doi:10.1021/tx300011w
PMCID: PMC3723381  PMID: 22332732
9.  Replication of the 2,6-Diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (MeFapy-dGuo) Adduct by Eukaryotic DNA Polymerases 
Chemical research in toxicology  2012;25(8):1652-1661.
N6-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8–29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability.
doi:10.1021/tx300113e
PMCID: PMC3682502  PMID: 22721435
10.  Replication Bypass of the Acrolein-Mediated Deoxyguanine DNA–Peptide Cross-links by DNA polymerases of the DinB Family 
Chemical research in toxicology  2008;21(10):1983-1990.
DNA–protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an α,β-unsaturated aldehyde, acrolein. DNA–protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA–peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA–peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or dodecylpeptide was covalently attached at the N2 position of guanine via an acrolein adduct, and replication bypass assays carried out with members of the DinB family of polymerases, human polymerase (pol)κ and Escherichia coli (E. coli) pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol κ was able to catalyze both the incorporation and extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all these substrates, suggesting that pol κ would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol κ, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA–peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA–peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N2-guanine linked DNA–peptide cross-links.
doi:10.1021/tx800174a
PMCID: PMC2673917  PMID: 18788757
11.  8-Oxoguanine DNA Glycosylase (OGG1) Deficiency Increases Susceptibility to Obesity and Metabolic Dysfunction 
PLoS ONE  2012;7(12):e51697.
Oxidative damage to DNA is mainly repaired via base excision repair, a pathway that is catalyzed by DNA glycosylases such as 8-oxoguanine DNA glycosylase (OGG1). While OGG1 has been implicated in maintaining genomic integrity and preventing tumorigenesis, we report a novel role for OGG1 in altering cellular and whole body energy homeostasis. OGG1-deficient (Ogg1−/−) mice have increased adiposity and hepatic steatosis following exposure to a high-fat diet (HFD), compared to wild-type (WT) animals. Ogg1−/− animals also have higher plasma insulin levels and impaired glucose tolerance upon HFD feeding, relative to WT counterparts. Analysis of energy expenditure revealed that HFD-fed Ogg1−/− mice have a higher resting VCO2 and consequently, an increased respiratory quotient during the resting phase, indicating a preference for carbohydrate metabolism over fat oxidation in these mice. Additionally, microarray and quantitative PCR analyses revealed that key genes of fatty acid oxidation, including carnitine palmitoyl transferase-1, and the integral transcriptional co-activator Pgc-1α were significantly downregulated in Ogg1−/− livers. Multiple genes involved in TCA cycle metabolism were also significantly reduced in livers of Ogg1−/− mice. Furthermore, hepatic glycogen stores were diminished, and fasting plasma ketones were significantly reduced in Ogg1−/− mice. Collectively, these data indicate that OGG1 deficiency alters cellular substrate metabolism, favoring a fat sparing phenotype, that results in increased susceptibility to obesity and related pathologies in Ogg1−/− mice.
doi:10.1371/journal.pone.0051697
PMCID: PMC3524114  PMID: 23284747
12.  A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ 
PLoS ONE  2012;7(10):e45032.
Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N2-dG DNA–DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.
doi:10.1371/journal.pone.0045032
PMCID: PMC3466269  PMID: 23056190
13.  Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site 
PLoS ONE  2012;7(2):e31377.
In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N–terminal amine.
doi:10.1371/journal.pone.0031377
PMCID: PMC3285167  PMID: 22384015
14.  Role of High-Fidelity Escherichia coli DNA Polymerase I in Replication Bypass of a Deoxyadenosine DNA-Peptide Cross-Link ▿  
Journal of Bacteriology  2011;193(15):3815-3821.
Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (N6-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past N6-dA-linked peptides. Based on these results, we hypothesized that another member of that family, Escherichia coli polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific N6-dA dodecylpeptide cross-link were created and utilized for in vitro DNA replication assays using E. coli DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using E. coli strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific N6-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past N6-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.
doi:10.1128/JB.01550-10
PMCID: PMC3147505  PMID: 21622737
15.  TAT-mediated delivery of a DNA repair enzyme to skin cells rapidly initiates repair of UV-induced DNA damage 
Ultraviolet (UV) light causes DNA damage in skin cells, leading to more than one million cases of non-melanoma skin cancer diagnosed annually in the United States. Although human cells possess a mechanism (Nucleotide Excision Repair, NER) to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated prior to repair of these photoproducts. While human cells have all the enzymes necessary to complete an alternate repair pathway, Base Excision Repair (BER), they lack a DNA glycosylase that can initiate BER of dipyrimidine photoproducts. Certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (TAT). Here, we demonstrate that the Cv-pdg-NLS-TAT protein was delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. These data suggest a potential strategy for prevention of human skin cancer.
doi:10.1038/jid.2010.300
PMCID: PMC3203208  PMID: 20927123
16.  Mutagenic potential of DNA–peptide crosslinks mediated by acrolein-derived DNA adducts 
Mutation research  2007;637(1-2):161-172.
Current data suggest that DNA–peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA–protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA–peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA–peptide crosslinks in mammalian cells. Site-specific DNA–peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N6 position of deoxyadenosine (dA) or the N2 position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (γ-hydroxypropano-dA or γ-hydroxypropano-dG, respectively). In order to evaluate potential of DNA–peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells, and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the γ-hydroxypropano-dA-mediated crosslink induced mutations at only ~ 0.4%. In contrast, replication bypass of the γ-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of ~ 8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at γ-hydroxypropano-dG caused an increase in mutation frequencies relative to γ-hydroxypropano-dG, similar modification of γ-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA–peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.
doi:10.1016/j.mrfmmm.2007.08.001
PMCID: PMC3181171  PMID: 17868748
17.  Modulation of the Turnover of Formamidopyrimidine DNA Glycosylase (Fpg) † 
Biochemistry  2006;45(23):7341-7346.
In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed post-incision turnover events. Previously, we identified a critical residue (His16) in the T4 pyrimidine dimer glycosylase (T4-Pdg) that when mutated, interferes with enzyme turnover [Meador et al. (2004) J Biol Chem 279, 3348–3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase-AP lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate, but had turnover rates comparable to wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate limiting step catalyzed by BER glycosylase-AP lyases.
doi:10.1021/bi052383p
PMCID: PMC3181173  PMID: 16752923
18.  DNA Cross-links Induced by trans-4-Hydroxynonenal 
trans-4-Hydroxynonenal (HNE) is a peroxidation product of ω-6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1,N2-dG adducts. The adduct of (6S,8R,11S) stereochemistry forms inter-strand N2-dG:N2-dG cross-links in the 5′-CpG-3′ sequence. It has been compared with the (6R,8S,11R) adduct, incorporated into 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTCGCTAGC)-3′, containing the 5′-CpG-3′ sequence (X = HNE-dG). Both adducts rearrange in DNA to N2-dG aldehydes. These aldehydes exist in equilibrium with diastereomeric cyclic hemiacetals, in which the latter predominate at equilibrium. These cyclic hemiacetals mask the aldehydes, explaining why DNA cross-linking is slow compared to related 1,N2-dG adducts formed by acrolein and crotonaldehyde. Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals are located within the minor groove. However, the (6S,8R,11S) cyclic hemiacetal orients in the 5′-direction, while the (6R,8S,11R) cyclic hemiacetal orients in the 3′-direction. The conformations of the diastereomeric N2-dG aldehydes, which are the reactive species involved in DNA cross-link formation, have been calculated using molecular mechanics methods. The (6S,8R,11S) aldehyde orients in the 5′-direction, while the (6R,8S,11R) aldehyde orients in the 3′-direction. This suggests a kinetic basis to explain, in part, why the (6S,8R,11S) HNE adduct forms interchain cross-links in the 5′-CpG-3′ sequence, whereas (6R,8S,11R) HNE adduct does not. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease.
doi:10.1002/em.20599
PMCID: PMC3140422  PMID: 20577992
19.  γ-Hydroxy-1,N2-propano-2′-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage 
Chemical Research in Toxicology  2011;24(7):1123-1133.
The γ-hydroxy-1,N2-propano-2′-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5′-d(GCTAGCXAGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (X = γ-OH-PdG). In the presence of excess peptide KWKK, 13C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA–KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N2-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited 15N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH3, suggesting that they existed, in part, as imine (Schiff base) linkages. However, 13C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA–KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. (2010) Biochemistry, 24, 6155−6164]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA–KWKK conjugates thermally stabilized duplex DNA. However, the DNA–KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5′-CpX-3′ sequence, the DNA–KWKK conjugates slowly converted to interstrand N2-dG:N2-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks.
doi:10.1021/tx200113n
PMCID: PMC3138414  PMID: 21561113
20.  The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration 
PLoS ONE  2011;6(3):e17862.
An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.
doi:10.1371/journal.pone.0017862
PMCID: PMC3063173  PMID: 21448280
21.  Evidence for the Involvement of DNA Repair Enzyme NEIL1 in Nucleotide Excision Repair of (5′R)- and (5′S)-8,5′-Cyclo-2′-deoxyadenosines† 
Biochemistry  2010;49(6):1053-1055.
The DNA repair enzyme NEIL1 is a DNA glycosylase that is involved in the first step of base excision repair (BER) of oxidatively induced DNA damage. NEIL1 exhibits a strong preference for excision of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA with no specificity for 8-hydroxyguanine (8-OH-Gua). In this study, we report on the significant accumulation of (5′R)-8,5′-cyclo-2′-deoxyadenosine (R-cdA) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA) in liver DNA of neil1−/− mice that were not exposed to exogenous oxidative stress, while no accumulation of these lesions was observed in liver DNA from control or ogg1−/− mice. Significant accumulation of FapyGua was detected in liver DNA of both neil1−/− and ogg1−/− mice, while 8-OH-Gua accumulated in ogg1−/− only. Since R-cdA and S-cdA contain an 8,5′-covalent bond between the base and sugar moieties, they cannot be repaired by BER. There is evidence that these lesions are repaired by nucleotide excision repair (NER). Since the accumulation of R-cdA and S-cdA in neil1−/− mice strongly points to the failure of their repair, these data suggest that NEIL1 is involved in NER of R-cdA and S-cdA. Further studies aimed at elucidating the mechanism of action of NEIL1 in NER are warranted.
doi:10.1021/bi902161f
PMCID: PMC2817919  PMID: 20067321
22.  Conformational Interconversion of the trans-4-Hydroxynonenal-Derived (6S,8R,11S) 1,N2-Deoxyguanosine Adduct When Mismatched with Deoxyadenosine in DNA 
Chemical research in toxicology  2009;22(1):187-200.
The (6S,8R,11S) 1,N2-HNE-dG adduct of trans-4-hydroxynonenal (HNE) was incorporated into the duplex 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTAGCTAGC)-3′ [X=(6S,8R,11S) HNE-dG], in which the lesion was mismatched opposite dA. The (6S,8R,11S) adduct maintained the ring-closed 1,N2-HNE-dG structure. This was in contrast to when this adduct was correctly paired with dC, conditions under which it underwent ring opening and re-arrangement to diastereomeric minor groove hemiacetals [Huang, H., Wang, H., Qi, N., Lloyd, R.S., Harris, T.M., Rizzo, C.J., & Stone, M.P. (2008) J. Am. Chem. Soc. 130, 10898–10906]. The (6S,8R,11S) adduct exhibited a syn/anti conformational equilibrium about the glycosyl bond. The syn conformation was predominant in acidic solution. Structural analysis of the syn conformation revealed that X7 formed a distorted base pair with the complementary protonated A18. The HNE moiety was located in the major groove. Structural perturbations were observed at the neighbor C6•G19 and A8•T17 base pairs. At basic pH, the anti conformation of X7 was the major species. At X7 the 1,N2-HNE-dG intercalated and displaced the complementary A18 in the 5′-direction, resulting in a bulge at the X7•A18 base pair. The HNE aliphatic chain was oriented towards the minor groove. The Watson-Crick hydrogen bonding of the neighboring A8•T17 base pair was also disrupted.
doi:10.1021/tx800320m
PMCID: PMC2645987  PMID: 19053179
23.  Interstrand DNA Cross-links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources 
Accounts of chemical research  2008;41(7):793-804.
Conspectus
Significant levels of the 1,N2-γ-hydroxypropano-dG adducts of the α,β-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposure. They yield interstrand DNA cross-links between guanines in the neighboring C•G and G•C base pairs located in 5′-CpG-3′ sequences, as a result of opening of the 1,N2-γ-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C•G and G•C base pairs located in 5′-CpG-3′ sequences, that maintain the Watson–Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N2-γ-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.
doi:10.1021/ar700246x
PMCID: PMC2785109  PMID: 18500830
24.  Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal 
Chemical Research in Toxicology  2009;22(5):759-778.
The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.
doi:10.1021/tx9000489
PMCID: PMC2685875  PMID: 19397281
25.  Uncoupling of nucleotide flipping and DNA bending by the T4-pyrimidine dimer DNA glycosylase+ 
Biochemistry  2006;45(47):14192-14200.
Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5′ thymine of a thymine-thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60° in the enzyme-substrate (ES) complex. To examine the kinetic details of the pre-catalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately measure nucleotide flipping and DNA bending. The fluorescent adenine base analog, 2-aminopurine (2-AP) placed opposite an abasic site analog, tetrahydrofuran, exhibited a 2.8 fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping, kon and koff presteady-state kinetic measurements were determined. DNA bending was measured by fluorescence resonance energy transfer using fluorescent donor/acceptor pairs located at the 5′ ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5 fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.
doi:10.1021/bi060802s
PMCID: PMC2673921  PMID: 17115714

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