Albena Kozekova

Interstrand DNA Cross-links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. 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,N(2)-gamma-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,N(2)-gamma-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.

Role for DNA Polymerase κ in the Processing of N2-N2-Guanine Interstrand Cross-links

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N2-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ′ and 3 ′ ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol ζ or a pol ζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.

Replication Bypass of the Acrolein-Mediated Deoxyguanine DNA- Peptide Cross-links by DNA polymerases of the DinB Family

DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-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 a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa 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 kappa, 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 N (2)-guanine-linked DNA-peptide cross-links.

DNA cross-link 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 interstrand 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. Environ. Mol. Mutagen., 2010.

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA–topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative α,β-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA–Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA–Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA–Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.

Stereospecific Formation of the (R)-γ-Hydroxytrimethylene Interstrand N2-dG:N2-dG Cross-Link Arising from the γ-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.

Minor Groove Orientation of the KWKK Peptide Tethered via the N-Terminal Amine to the Acrolein-Derived 1,N2-γ-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 N2-dG amine positioned in 5′-d(GCTAGCXAGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (X = N2-dG−trimethylene link−KWKK). This linkage is a surrogate for the reversible linkage formed by the γ-OH-1,N2-propanodeoxyguanosine (γ-OH-PdG) adduct. This conjugated KWKK stabilizes the DNA. Amino acids K26, W27, K28, and K29 are in the minor groove. The W27 indolyl group does not intercalate into the DNA. The G7N2 amine and the K26 N-terminal amine nitrogens are in the trans configuration with respect to the Cα or Cγ of the trimethylene tether, respectively. The structure of this DNA−KWKK conjugate is discussed in the context of its biological processing.

Translesion DNA Synthesis by Human DNA Polymerase η on Templates Containing a Pyrimidopurinone Deoxyguanosine Adduct, 3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one

M1dG (3-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M1dG to dT and M1dG to dA) and frameshift mutations. M1dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M1dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase η to synthesize DNA across from M1dG was studied. M1dG partially blocked DNA synthesis by polymerase η. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M1dG lesion (800-fold less efficient than opposite dG). Extension from M1dG·dC was equally as efficient as from control primer-templates (dG·dC). dATP insertion opposite M1dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M1dG·dA was 20-fold less efficient than dG·dC. The sequences of full-length human DNA polymerase η bypass products of M1dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M1dG or −1 frameshifts at the lesion site (31%). Human DNA polymerase η bypass may lead to M1dG to dT and frameshift but likely not M1dG to dA mutations during DNA replication.

Formation of a N2-dG:N2-dG carbinolamine DNA cross-link by the trans-4-hydroxynonenal-derived (6S,8R,11S) 1,N2-dG adduct.

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N2-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6S,8R,11S) diastereomer forms a N2-dG:N2-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687–5700]. We refined its structure in 5′-d(G1C2T3A4G5C6X7A8G9T10C11C12)-3′·5′-d(G13G14A15C16T17C18Y19C20T21A22G23C24)-3′ [X7 is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N2-dG adduct, and Y19 is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N2-dG adduct; the cross-link is in the 5′-CpG-3′ sequence]. Introduction of 13C at the C8 carbon of the cross-link revealed one 13C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y19 H1′, C20 H1′, and C20 H4′, orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y19 H1′, orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y19N2H and X7 N1H protons, respectively. A strong H8→H11 NOE and no 3J(13C→H) coupling for the 13C8–O–C11–H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N2-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X7N2 and Y19N2 atoms were in the gauche conformation with respect to the linkage, maintaining Watson–Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C20O2 of the 5′-neighbor base pair G5·C20 and O11H with C18O2 of X7·C18. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.

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.