Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release.

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 µs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the "closed-open" transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

Naphthablins B and C, Meroterpenoids Identified from the Marine Sediment-Derived Streptomyces sp. CP26-58 Using HeLa Cell-Based Cytological Profiling.

HeLa cell-based cytological profiling (CP) was applied to an extract library of marine sediment-derived actinomycetes to discover new cytotoxic secondary metabolites. Among the hit strains, Streptomyces sp. CP26-58 was selected for further investigation to identify its cytotoxic metabolites. CP revealed that the known ionophore tetronasin (1) was responsible for the cytotoxic effect found in the extract. Furthermore, three naphthoquinone meroterpenoids, naphthablin A (2) and two new derivatives designated as naphthablins B (3) and C (4), were isolated from other cytotoxic fractions. The structures of the new compounds were elucidated based on analysis of their HRESIMS and comprehensive NMR data. The absolute configurations of the new compounds were deduced by simulating ECD spectra and calculating potential energies for the model compounds using density function theory (DFT) calculations. Compound 1 showed a significant cytotoxic effect against HeLa cells with an IC50 value of 0.23 µM, and CP successfully clustered 1 with calcium ionophores.

Cadmium(II) inhibition of human uracil-DNA glycosylase by catalytic water supplantation.

Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 µM. Computational analyses based on 2 µs equilibrium, 1.6 µs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases.

Engineering trypsin for inhibitor resistance.

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

Evaluation of benzoic acid derivatives as sirtuin inhibitors.

Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p) inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured SIRT1 inhibitory activities.

Creation of an HDAC-based yeast screening method for evaluation of marine-derived actinomycetes: discovery of streptosetin A.

A histone deacetylase (HDAC)-based yeast assay employing a URA3 reporter gene was applied as a primary screen to evaluate a marine-derived actinomycete extract library and identify human class III HDAC (SIRT) inhibitors. On the basis of the bioassay-guided purification, a new compound designated as streptosetin A (1) was obtained from one of the active strains identified through the yeast assay. The gross structure of the new compound was elucidated from the 1D and 2D NMR data. The absolute stereostructure of 1 was determined based on X-ray crystal structure analysis and simulation of ECD spectra using time-dependent density functional theory calculations. This compound showed weak inhibitory activity against yeast Sir2p and human SIRT1 and SIRT2.

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants.

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 µs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.

Protein dynamics via computational microscope.

The purpose of this overview is to provide a concise introduction to the methodology and current advances in molecular dynamics (MD) simulations. MD simulations emerged as a powerful and popular tool to study dynamic behavior of proteins and macromolecule complexes at the atomic resolution. This approach can extend static structural data, such as X-ray crystallography, into dynamic domains with realistic timescales (up to millisecond) and high precision, therefore becoming a veritable computational microscope. This perspective covers current advances and methodology in the simulation of protein folding and drug design as illustrated by several important published examples. Overall, recent progress in the simulation field points to the direction that MD will have significant impact on molecular biology and pharmaceutical science.

Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA.

Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N(2)-(4-Hydroxyphenyl)-2'-deoxyguanosine (N(2)-4-HOPh-dG) is the principal adduct identified in vivo by (32)P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N(2)-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N(2)-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson-Crick hydrogen bonds are present within the N(2)-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N(2)-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

Substrate specificity of human thymine-DNA glycosylase on exocyclic cytosine adducts.

The environmental carcinogen glycidaldehyde (GDA) and therapeutic chloroethylnitrosoureas (CNUs) can form hydroxymethyl etheno and ring-saturated ethano bases, respectively. The mutagenic potential of these adducts relies on their miscoding properties and repair efficiency. In this work, the ability of human thymine-DNA glycosylase (TDG) to excise 8-(hydroxymethyl)-3,N(4)-ethenocytosine (8-hm-varepsilonC) and 3,N(4)-ethanocytosine (EC) was investigated and compared with varepsilonC, a known substrate for TDG. When tested using defined oligonucleotides containing a single adduct, TDG is able to excise 8-hm-varepsilonC but not EC. The 8-hm-varepsilonC activity mainly depends on guanine pairing with the adduct. TDG removes 8-hm-varepsilonC less efficiently than varepsilonC but its activity can be significantly enhanced by human AP endonuclease 1 (APE1), a downstream enzyme in the base excision repair. TDG did not show any detectable activity toward EC when placed in various neighboring sequences, including the 5'-CpG site. Molecular modeling revealed a possible steric clash between the non-planar EC exocyclic ring and residue Asn 191 within the TDG active site, which could account for the lack of TDG activity toward EC. TDG was not active against the bulkier exocyclic adduct 3,N(4)-benzethenocytosine, nor the two adenine derivatives with same modifications as the cytosine derivatives, 7-hm-varepsilonA and EA. These findings expand the TDG substrate range and aid in understanding the structural requirements for TDG substrate specificity.

Metal inhibition of human N-methylpurine-DNA glycosylase activity in base excision repair.

Cadmium (Cd2+), nickel (Ni2+) and cobalt (Co2+) are human and/or animal carcinogens. Zinc (Zn2+) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd2+, Ni2+, and Zn2+ can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (varepsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1,000 microM, both Cd2+ and Zn2+ showed metal-dependent inhibition of the MPG catalytic activity. Ni2+ also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co2+ and Mg2+ did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the varepsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd2+, Zn2+, and Ni2+ at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn2+ showed that the MPG active site has a potential binding site for Zn2+, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

Water transport in AQP0 aquaporin: molecular dynamics studies.

A double lipid bilayer structure containing opposing tetramers of AQP0 aquaporin, in contact through extracellular face loop regions, was recently modeled using an intermediate-resolution map obtained by electron crystallographic methods. The pores of these water channels were found to be critically narrow in three regions and subsequently interpreted to be those of a closed state of the channel. The subsequent determination of a high-resolution AQP0 tetramer structure by X-ray crystallographic methods yielded a pore model featuring two of the three constrictions as noted in the EM work and water molecules within the channel pore. The extracellular-side constriction region of this AQP0 structure was significantly larger than that of the EM-based model and similar to that of the highly water permeable AQP1. The X-ray-based study of AQP0 however could not ascertain if the water molecules found in the pore were the result of water entering from one or both ends of the channel, nor whether water could freely pass through all constriction points. Additionally, this X-ray-based structure could not provide an answer to the question of whether the double lipid bilayer configuration of AQP0 could functionally maintain a water impermeable state of the channel. To address these questions we conducted molecular dynamics simulations to compare the time-dependent behavior of the AQP0 and AQP1 channels within lipid bilayers. The simulations demonstrate that AQP0, in single or double lipid bilayers, is not closed to water transport and that thermal motions of critical side-chains are sufficient to facilitate the movement of water past any of its constriction regions. These motional requirements do however lead to significant free energy barriers and help explain physiological observations that found water permeability in AQP0 to be substantially lower than in the AQP1 pore.

Alkylpurine-DNA-N-glycosylase excision of 7-(hydroxymethyl)-1,N6-ethenoadenine, a glycidaldehyde-derived DNA adduct.

Glycidaldehyde (GDA) is a bifunctional alkylating agent that has been shown to be mutagenic in vitro and carcinogenic in rodents. However, the molecular mechanism by which it exerts these effects is not established. GDA is capable of forming exocyclic hydroxymethyl-substituted etheno adducts on base residues in vitro. One of them, 7-(hydroxymethyl)-1,N6-ethenoadenine (7-hm-epsilonA), was identified as the principal adduct in mouse skin treated with GDA or a glycidyl ether. In this work, using defined oligonucleotides containing a site-specific 7-hm-epsilonA, the human and mouse alkylpurine-DNA-N-glycosylases (APNGs), responsible for the removal of the analogous 1,N6-ethenoadenine (epsilonA) adduct, are shown to recognize and excise 7-hm-epsilonA. Such an activity can be significantly modulated by both 5' neighboring and opposite sequence contexts. The efficiency of human or mouse APNG for excision of 7-hm-epsilonA is about half that, or similar to the excision of epsilonA, respectively. When human or mouse cell-free extracts were tested, however, the extent of 7-hm-epsilonA excision is dramatically lower than that for epsilonA, suggesting that, in the crude extracts, the APNG activities toward these two adducts are differentially affected. Using cell-free extracts from APNG deficient mice, this enzyme is shown to be the primary glycosylase excising 7-hm-epsilonA. A structural approach, using molecular modeling, was employed to examine how the structure of the 7-hm-epsilonA adduct affects DNA conformation, as compared to the epsilonA adduct. These novel substrate specificities could have both biological and structural implications.

The p-benzoquinone DNA adducts derived from benzene are highly mutagenic.

Benzene is a human leukemia carcinogen, resulting from its cellular metabolism. A major benzene metabolite is p-benzoquinone (pBQ), which can damage DNA by forming the exocyclic base adducts pBQ-dC, pBQ-dA, and pBQ-dG in vitro. To gain insights into the role of pBQ in benzene genotoxicity, we examined in vitro translesion synthesis and in vivo mutagenesis of these pBQ adducts. Purified REV1 and Polkappa were essentially incapable of translesion synthesis in response to the pBQ adducts. Opposite pBQ-dA and pBQ-dC, purified human Poliota was capable of error-prone nucleotide insertion, but was unable to perform extension synthesis. Error-prone translesion synthesis was observed with Poleta. However, DNA synthesis largely stopped opposite the lesion. Consistent with in vitro results, replication of site-specifically damaged plasmids was strongly inhibited by pBQ adducts in yeast cells, which depended on both Polzeta and Poleta. In wild-type cells, the majority of translesion products were deletions at the site of damage, accounting for 91%, 90%, and 76% for pBQ-dA, pBQ-dG, and pBQ-dC, respectively. These results show that the pBQ-dC, pBQ-dA, and pBQ-dG adducts are strong blocking lesions, and are highly mutagenic by predominantly inducing deletion mutations. These results are consistent with the lesion structures predicted by molecular dynamics simulation. Our results led to the following model. Translesion synthesis normally occurs by directly copying the lesion site through base insertion and extension synthesis. When the lesion becomes incompatible in accommodating a base opposite the lesion in DNA, translesion synthesis occurs by a less efficient lesion loop-out mechanism, resulting in avoiding copying the damaged base and leading to deletion.

Chloroethylnitrosourea-derived ethano cytosine and adenine adducts are substrates for Escherichia coli glycosylases excising analogous etheno adducts.

Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N4-ethanocytosine (EC) and 1,N6-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N4-ethenocytosine (epsilonC) and AlkA releases 1,N6-ethenoadenine (epsilonA). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a approximately 20-fold lower excision activity toward EC or EA than that toward their structurally analogous epsilonC or epsilonA adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the epsilonC base, which could account for the lower EC activity observed in this study.

Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C.

The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site.

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea.

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.

Structural insights by molecular dynamics simulations into differential repair efficiency for ethano-A versus etheno-A adducts by the human alkylpurine-DNA N-glycosylase.

1,N6-ethenoadenine adducts (epsilonA) are formed by known environmental carcinogens and found to be removed by human alkylpurine-DNA N-glycosylase (APNG). 1,N6-ethanoadenine (EA) adducts differ from epsilonA by change of a double bond to a single bond in the 5-member exocyclic ring and are formed by chloroethyl nitrosoureas, which are used in cancer therapy. In this work, using purified recombinant human APNG, we show that EA is a substrate for the enzyme. However, the excision efficiency of EA was 65-fold lower than that of epsilonA. Molecular dynamics simulation produced similar structural motifs for epsilonA and EA when incorporated into a DNA duplex, suggesting that there are no specific conformational features in the DNA duplex which can account for the differences in repair efficiency. However, when EA was modeled into the APNG active site, based on the APNG/epsilonA-DNA crystallographic coordinates, in structures produced by 2 ns molecular dynamics simulation, we observed weakening in the stacking interaction between EA and aromatic side chains of the key amino acids in the active site. In contrast, the planar epsilonA is better stacked at the enzyme active site. We propose that the observed destabilization of the EA adduct at the active site, such as reduced stacking interactions, could account for the biochemically observed weaker recognition of EA by APNG as compared to epsilonA.

Novel activity of Escherichia coli mismatch uracil-DNA glycosylase (Mug) excising 8-(hydroxymethyl)-3,N4-ethenocytosine, a potential product resulting from glycidaldehyde reaction.

Glycidaldehyde is an industrial chemical which has been shown to be genotoxic in in vitro experiments and carcinogenic in rodent studies. It is a bifunctional alkylating agent capable of reacting with DNA to form exocyclic hydroxymethyl-substituted ethenobases. In this work, 8-(hydroxymethyl)-3,N4-etheno-2'-deoxycytidine (8-HM-epsilondC), a potential nucleoside derivative of glycidaldehyde, was synthesized using phosphoramidite chemistry and site-specifically incorporated into a defined 25-mer oligodeoxynucleotide. The 8-HM-epsilonC adduct is structurally related to 3,N4-ethenocytosine (epsilonC), a product of reaction with vinyl chloride or through lipid peroxidation. In Escherichia coli, epsilonC has been shown previously to be a primary substrate for the mismatch uracil-DNA glycosylase (Mug). In this study, we report that the same glycosylase also acts on 8-HM-epsilonC in an oligonucleotide duplex. The enzyme binds to the 8-HM-epsilonC-oligonucleotide to a similar extent as the epsilonC-oligonucleotide. The Mug excision activity toward 8-HM-epsilonC is approximately 2.5-fold lower than that toward the epsilonC substrate. Both activities can be stimulated up to approximately 2-fold higher by the addition of E. coli endonuclease IV. These two adducts, when mispaired with normal bases, were all excised from DNA by Mug with similar efficiencies. Structural studies using molecular simulations showed similar adjustment and hydrogen bonding pattern for both 8-HM-epsilonC*G and epsilonC*G pairs in oligomer duplexes. We believe that these findings may have biological and structural implications in defining the role of 8-HM-epsilonC in glycosylase recognition/repair.

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases.

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.