Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

Ring-opening of the γ-OH-PdG adduct promotes error-free bypass by the Sulfolobus solfataricus DNA polymerase Dpo4.

Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2'-deoxy-ß-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 N(2)-(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.

Deoxyguanosine forms a bis-adduct with E,E-muconaldehyde, an oxidative metabolite of benzene: implications for the carcinogenicity of benzene.

Benzene is employed in large quantities in the chemical industry and is an ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans, but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found that the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in the formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH(4). The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA, but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, the imidazole NH of histidine, and N termini of peptides with the dGuo-muconaldehyde monoadduct.

1,N2-Etheno-2'-deoxyguanosine adopts the syn conformation about the glycosyl bond when mismatched with deoxyadenosine.

The oligodeoxynucleotide 5'-CGCATXGAATCC-3'·5'-GGATTCAATGCG-3' containing 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdG) opposite deoxyadenosine (named the 1,N(2)-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,N(2)-εdG by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and by Escherichia coli polymerases pol I exo(-) and pol II exo(-). At pH 5.2, the T(m) of this duplex was increased by 3 °C as compared to the duplex in which the 1,N(2)-εdG lesion is opposite dC, and it was increased by 2 °C compared to the duplex in which guanine is opposite dA (the dG·dA duplex). A strong NOE between the 1,N(2)-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A(20) H2 proton and the mismatched A(19) H2 proton from the complementary strand, establish that 1,N(2)-εdG rotated about the glycosyl bond from the anti to the syn conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T(5) CH(3). A strong NOE between A(20) H2 and A(19) H2 protons established that A(19), opposite to 1,N(2)-εdG, adopted the anti conformation and was directed toward the helix. The downfield shifts of the A(19) amino protons suggested protonation of dA. Thus, the protonated 1,N(2)-εdG·dA base pair was stabilized by hydrogen bonds between 1,N(2)-εdG N1 and A(19) N1H(+) and between 1,N(2)-εdG O(9) and A(19)N(6)H. The broad imino proton resonances for the 5'- and 3'-flanking bases suggested that both neighboring base pairs were perturbed. The increased stability of the 1,N(2)-εdG·dA base pair, compared to that of the 1,N(2)-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,N(2)-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,N(2)-εdG.

Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal.

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,ß-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 µg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

Minor groove orientation of the KWKK peptide tethered via the N-terminal amine to the acrolein-derived 1,N2-gamma-hydroxypropanodeoxyguanosine lesion with a trimethylene linkage.

DNA-protein conjugates are potentially repaired via proteolytic digestion to DNA-peptide conjugates. The latter have been modeled with the amino-terminal lysine of the peptide KWKK conjugated via a trimethylene linkage to the N(2)-dG amine positioned in 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3' (X = N(2)-dG-trimethylene link-KWKK). This linkage is a surrogate for the reversible linkage formed by the gamma-OH-1,N(2)-propanodeoxyguanosine (gamma-OH-PdG) adduct. This conjugated KWKK stabilizes the DNA. Amino acids K(26), W(27), K(28), and K(29) are in the minor groove. The W(27) indolyl group does not intercalate into the DNA. The G(7) N(2) amine and the K(26) N-terminal amine nitrogens are in the trans configuration with respect to the C(alpha) or C(gamma) of the trimethylene tether, respectively. The structure of this DNA-KWKK conjugate is discussed in the context of its biological processing.

Comparison of the in vitro replication of the 7-(2-oxoheptyl)-1,N2-etheno-2'-deoxyguanosine and 1,N2-etheno-2'-deoxyguanosine lesions by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4).

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to that of the 1,N(2)-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in 5'-TXG-3' local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5'-CXG-3' local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N(2)-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5'-TXG-3' sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pK(a) for this transition was lower than that observed for the 1,N(2)-epsilon-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pK(a) value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N(2)-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.

DNA cross-link induced by trans-4-hydroxynonenal.

Trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1,N(2)-dG adducts. The adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-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 N(2)-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,N(2)-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 N(2)-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.

Structure of the 1,N(2)-etheno-2'-deoxyguanosine lesion in the 3'-G(epsilon dG)T-5' sequence opposite a one-base deletion.

The structure of the 1,N(2)-ethenodeoxyguanosine lesion (1,N(2)-epsilondG) has been characterized in 5'-d(CGCATXGAATCC)-3'.5'-d(GGATTCATGCG)-3' (X = 1,N(2)-epsilondG), in which there is no dC opposite the lesion. This duplex (named the 1-BD duplex) models the product of translesion bypass of 1,N(2)-epsilondG by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) [Zang, H., Goodenough, A. K., Choi, J. Y., Irimia, A., Loukachevitch, L. V., Kozekov, I. D., Angel, K. C., Rizzo, C. J., Egli, M., and Guengerich, F. P. (2005) J. Biol. Chem. 280, 29750-29764], leading to a one-base deletion. The T(m) of this duplex is 6 degrees C higher than that of the duplex in which dC is present opposite the 1,N(2)-epsilondG lesion and 8 degrees C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,N(2)-epsilondG imidazole and deoxyribose H1' protons and between the 1,N(2)-epsilondG etheno H6 and H7 protons and DNA protons establishes that 1,N(2)-epsilondG adopts the anti conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,N(2)-epsilondG H6 and H7 etheno protons shift upfield relative to the monomer 1,N(2)-epsilondG, attributed to ring current shielding, consistent with their intrahelical location. NMR data reveal that Watson-Crick base pairing is maintained at both the 5' and 3' neighbor base pairs. The structure of the 1-BD duplex has been refined using molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints. The increased stability of the 1,N(2)-epsilondG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,N(2)-epsilondG lesion by the Dpo4 polymerase, suggesting that stabilization of this bulged intermediate may be significant with regard to the biological processing of the lesion.

Novel enzymatic function of DNA polymerase nu in translesion DNA synthesis past major groove DNA-peptide and DNA-DNA cross-links.

DNA polymerase nu (POLN or pol nu) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol nu, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol nu catalyzed efficient and high fidelity TLS past peptides linked to N(6)-dA via a reduced Schiff base linkage with a gamma-hydroxypropano-dA. Additionally, pol nu could bypass DNA interstrand cross-links with linkage between N(6)-dAs in complementary DNA strands. However, the chemically identical DNA--peptide and DNA interstrand cross-links completely blocked pol nu when they were located in the minor groove via a N(2)-dG linkage. Furthermore, we showed that pol nu incorporated a nucleotide opposite the 1,N(6)-etheno-dA (epsilondA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol nu bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol nu may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA-protein and DNA-DNA cross-links.

Checkpoint signaling from a single DNA interstrand crosslink.

DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.

Steric and electrostatic effects at the C2 atom substituent influence replication and miscoding of the DNA deamination product deoxyxanthosine and analogs by DNA polymerases.

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.

Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N2,N2-dimethyl (Me2)-substituted guanine (N2,N2-Me2G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N2,N2-Me2G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N2,N2-Me2G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N2,N2-Me2G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3'-terminal dideoxycytosine (Cdd) opposite template N2,N2-Me2G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N2,N2-Me2G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N2,N2-Me2G, with a second water-mediated hydrogen bond between the N-3 atom of Cdd and the O-6 atom of N2,N2-Me2G. A crystal structure of Dpo4 with dTTP opposite template N2,N2-Me2G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N2,N2-Me2G compared with mono-substituted N2-alkyl G adducts.

Stereospecific formation of the (R)-gamma-hydroxytrimethylene interstrand N2-dG:N2-dG cross-link arising from the gamma-OH-1,N2-propano-2'-deoxyguanosine adduct in the 5'-CpG-3' DNA sequence.

Acrolein reacts with dG to form hydroxylated 1,N(2)-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2a] purin-10(3H)-ones, commonly referred to as the gamma-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the N(2)-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence. Here we report the structure of the stereochemically favored (R)-gamma-hydroxytrimethylene N(2)-dG:N(2)-dG interstrand DNA cross-link in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3' x 5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' (X(7) is the dG linked to the alpha-carbon of the carbinolamine linkage, and Y(19) is the dG linked to the gamma-carbon of the carbinolamine linkage; the cross-link is in the 5'-CpG-3' sequence). The structure was characterized using isotope-edited (15)N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X(7) or Y(19) were (15)N-labeled. Analyses of NOE intensities involving Y(19) N(2)H indicated that the (R)-gamma-hydroxytrimethylene linkage was the major cross-link species, constituting 80-90% of the cross-link. The X(7) and Y(19) imino resonances were observed at 65 degrees C. Additionally, for the 5'-neighbor base pair G(5) x C(20), the G(5) imino resonance remained sharp at 55 degrees C but broadened at 65 degrees C. In contrast, for the 3'-neighbor A(8) x T(17) base pair, the T(17) imino resonance was severely broadened at 55 degrees C. Structural refinement using NOE distance restraints obtained from isotope-edited (15)N NOESY-HSQC data indicated that the (R)-gamma-hydroxytrimethylene linkage maintained the C(6) x Y(19) and X(7) x C(18) base pairs with minimal structural perturbations. The (R)-gamma-hydroxytrimethylene linkage was located in the minor groove. The X(7) N(2) and Y(19) N(2) atoms were in the gauche conformation with respect to the linkage, which maintained Watson-Crick hydrogen bonding of the cross-linked base pairs. The anti conformation of the hydroxyl group with respect to C(alpha) of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C(20) O(2) located in the 5'-neighboring base pair G(5) x C(20). The formation of this hydrogen bond may, in part, explain the thermal stability of this carbinolamine interstrand cross-link and the stereochemical preference for the (R) configuration of the cross-link.

Chemistry and biology of DNA containing 1,N(2)-deoxyguanosine adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxynonenal.

The alpha,beta-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 N(2)-amine to give N(2)-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N(2)-dG exocyclic products. The 1,N(2)-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N(2)-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N(2)-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,N(2)-dG exocyclic lesions undergo ring opening to the corresponding N(2)-(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 eta, pol iota, and pol kappa. It also can be accomplished by a combination of Rev1 and pol zeta acting sequentially. However, efficient nucleotide insertion opposite the 1,N(2)-dG ring-closed adducts can be carried out only by pol iota and Rev1, two DNA polymerases that do not rely on the Watson-Crick pairing to recognize the template base. The N(2)-(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 N(2)-dG:N(2)-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 N(2)-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol kappa in human, are implicated in error-free bypass of model acrolein-mediated N(2)-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.

Oxidation and glycolytic cleavage of etheno and propano DNA base adducts.

Non-invasive strategies for the analysis of endogenous DNA damage are of interest for the purpose of monitoring genomic exposure to biologically produced chemicals. We have focused our research on the biological processing of DNA adducts and how this may impact the observed products in biological matrixes. Preliminary research has revealed that pyrimidopurinone DNA adducts are subject to enzymatic oxidation in vitro and in vivo and that base adducts are better substrates for oxidation than the corresponding 2'-deoxynucleosides. We tested the possibility that structurally similar exocyclic base adducts may be good candidates for enzymatic oxidation in vitro. We investigated the in vitro oxidation of several endogenously occurring etheno adducts [1,N(2)-epsilon-guanine (1,N(2)-epsilon-Gua), N(2),3-epsilon-Gua, heptanone-1,N(2)-epsilon-Gua, 1,N(6)-epsilon-adenine (1,N(6)-epsilon-Ade), and 3,N(4)-epsilon-cytosine (3,N(4)-epsilon-Cyt)] and their corresponding 2'-deoxynucleosides. Both 1,N(2)-epsilon-Gua and heptanone-1,N(2)-epsilon-Gua were substrates for enzymatic oxidation in rat liver cytosol; heteronuclear NMR experiments revealed that oxidation occurred on the imidazole ring of each substrate. In contrast, the partially or fully saturated pyrimidopurinone analogues [i.e., 5,6-dihydro-M(1)G and 1,N(2)-propanoguanine (PGua)] and their 2'-deoxynucleoside derivatives were not oxidized. The 2'-deoxynucleoside adducts, 1,N(2)-epsilon-dG and 1,N(6)-epsilon-dA, underwent glycolytic cleavage in rat liver cytosol. Together, these data suggest that multiple exocyclic adducts undergo oxidation and glycolytic cleavage in vitro in rat liver cytosol, in some instances in succession. These multiple pathways of biotransformation produce an array of products. Thus, the biotransformation of exocyclic adducts may lead to an additional class of biomarkers suitable for use in animal and human studies.

Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts.

In contrast to replicative DNA polymerases, Sulfolobus solfataricus Dpo4 showed a limited decrease in catalytic efficiency (k(cat)/Km) for insertion of dCTP opposite a series of N2-alkylguanine templates of increasing size from (methyl (Me) to (9-anthracenyl)-Me (Anth)). Fidelity was maintained with increasing size up to (2-naphthyl)-Me (Naph). The catalytic efficiency increased slightly going from the N2-NaphG to the N2-AnthG substrate, at the cost of fidelity. Pre-steady-state kinetic bursts were observed for dCTP incorporation throughout the series (N2-MeG to N2-AnthG), with a decrease in the burst amplitude and k(pol), the rate of single-turnover incorporation. The pre-steady-state kinetic courses with G and all of the six N2-alkyl G adducts could be fit to a general DNA polymerase scheme to which was added an inactive complex in equilibrium with the active ternary Dpo4.DNA.dNTP complex, and only the rates of equilibrium with the inactive complex and phosphodiester bond formation were altered. Two crystal structures of Dpo4 with a template N2-NaphG (in a post-insertion register opposite a 3'-terminal C in the primer) were solved. One showed N2-NaphG in a syn conformation, with the naphthyl group located between the template and the Dpo4 "little finger" domain. The Hoogsteen face was within hydrogen bonding distance of the N4 atoms of the cytosine opposite N2-NaphG and the cytosine at the -2 position. The second structure showed N2-Naph G in an anti conformation with the primer terminus largely disordered. Collectively these results explain the versatility of Dpo4 in bypassing bulky G lesions.

Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

A two-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS3 scan mode. Buccal cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido[2,3-b]indole (AalphaC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AalphaC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 108 unmodified DNA bases, when 10 microg of DNA is employed for the assay.

Site-specific synthesis and characterization of oligonucleotides containing an N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion, the ring-opened product from N7-methylation of deoxyguanosine.

A phosphoramidite reagent of N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-1,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesions was synthesized in four steps from 2'-deoxyguanosine. Fapy nucleosides can rearrange to the pyranose form when the 5'-hydroxyl group is unprotected. The phosphoramidite was incorporated into oligonucleotides using solid-phase synthesis by adjusting the deprotection time for removal of the 5'-dimethoxytrityl group of the MeFapy-dGuo nucleotide, thereby minimizing its rearrangement to the ribopyranose. The furanose and pyranose forms were differentiated by a series of two-dimensional NMR experiments.