Formation of a <i>N</i>²-dG:<i>N</i>²-dG Carbinolamine DNA Cross-link by the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG Adduct

Michael addition of <i>trans</i>-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,<i>N</i>²-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6<i>S</i>,8<i>R</i>,11<i>S</i>) diastereomer forms a <i>N</i>²-dG:<i>N</i>²-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 5687–5700]. We refined its structure in 5′-d(G¹C²T³A⁴G⁵C⁶<u>X</u>⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸<u>Y</u>¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ [X⁷ is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct, and Y¹⁹ is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-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 (6<i>S</i>,8<i>R</i>,11<i>S</i>) 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 Y¹⁹ <i>N</i>²H and X⁷ N1H protons, respectively. A strong H8→H11 NOE and no ³<i>J</i>(¹³C→H) coupling for the ¹³C8–O–C11–H11 eliminated the tetrahydrofuran species derived from the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) carbinolamine linkage and the HNE side chain were located in the minor groove. The X⁷ <i>N</i>² and Y¹⁹ <i>N</i>² 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 C²⁰ <i>O</i>² of the 5′-neighbor base pair G⁵·C²⁰ and O11H with C¹⁸ <i>O</i>² of X⁷·C¹⁸. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration

Replication Bypass of the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>²-Deoxyguanosine DNA Adduct by the <i>Sulfolobus solfataricus</i> DNA Polymerase IV

<i>trans</i>-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the <i>N</i>²-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,<i>N</i>²-dGuo (1,<i>N</i>²-HNE-dGuo) adducts. The (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct was incorporated into the 18-mer templates 5′-d­(TCAT<u>X</u>GAATCCTTCCCCC)-3′ and d­(TCAC<u>X</u>GAATCCTTCCCCC)-3′, where X = (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct. These differed in the identity of the template 5′-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5′-d­(GGGGGAAGGATTC)-3′ or a 14-mer primer 5′-d­(GGGGGAAGGATTCC)-3′. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair. The <i>Sulfolobus solfataricus</i> P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct in a sequence-specific manner. If the template 5′-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5′-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5′-neighbor base is dThy. When presented with a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>²-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson–Crick pairing opposite the template 5′-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair, ring opening of the adduct to the corresponding <i>N</i>²-dGuo aldehyde species occurred. This allowed Watson–Crick base pairing at the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair

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

M₁dG (3-(2′-deoxy-β-d-<i>erythro</i>-pentofuranosyl)pyrimido[1,2-<i>a</i>]purin-10(3<i>H</i>)-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 <i>Escherichia coli</i> 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

Minor Groove Orientation of the KWKK Peptide Tethered via the N-Terminal Amine to the Acrolein-Derived 1,<i>N</i>²-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 <i>N</i>²-dG amine positioned in 5′-d(GCTAGC<u>X</u>AGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (<u>X</u> = <i>N</i>²-dG−trimethylene link−KWKK). This linkage is a surrogate for the reversible linkage formed by the γ-OH-1,<i>N</i>²-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 G⁷ <i>N</i>² amine and the K²⁶ N-terminal amine nitrogens are in the <i>trans</i> configuration with respect to the C_α or C_γ of the trimethylene tether, respectively. The structure of this DNA−KWKK conjugate is discussed in the context of its biological processing

γ-Hydroxy-1,<i>N</i>^<i>2</i>-propano-2′-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage

The γ-hydroxy-1,<i>N</i>^<i>2</i>-propano-2′-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5′-d(GCTAGC<u>X</u>AGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (<u>X</u> = 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 <i>N</i>^<i>2</i>-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, 49, 6155−6164]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the <i>S</i>-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 <i>N</i>²-dG:<i>N</i>²-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks

Stereospecific Formation of the (<i>R</i>)-γ-Hydroxytrimethylene Interstrand <i>N</i>²-dG:<i>N</i>²-dG Cross-Link Arising from the γ-OH-1,<i>N</i>²-Propano-2′-deoxyguanosine Adduct in the 5′-CpG-3′ DNA Sequence

Acrolein reacts with dG to form hydroxylated 1,<i>N</i>²-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-β-d-<i>erythro</i>-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2<i>a</i>] purin-10(3<i>H</i>)-ones, commonly referred to as the γ-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the <i>N</i>²-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand <i>N</i>²-dG:<i>N</i>²-dG cross-links in the 5′-CpG-3′ sequence. Here we report the structure of the stereochemically favored (<i>R</i>)-γ-hydroxytrimethylene <i>N</i>²-dG:<i>N</i>²-dG interstrand DNA cross-link in 5′-d(G¹C²T³A⁴G⁵C⁶X⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸Y¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ (X⁷ is the dG linked to the α-carbon of the carbinolamine linkage, and Y¹⁹ is the dG linked to the γ-carbon of the carbinolamine linkage; the cross-link is in the 5′-CpG-3′ sequence). The structure was characterized using isotope-edited ¹⁵N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X⁷ or Y¹⁹ were ¹⁵N-labeled. Analyses of NOE intensities involving Y¹⁹ <i>N</i>²H indicated that the (<i>R</i>)-γ-hydroxytrimethylene linkage was the major cross-link species, constituting 80−90% of the cross-link. The X⁷ and Y¹⁹ imino resonances were observed at 65 °C. Additionally, for the 5′-neighbor base pair G⁵·C²⁰, the G⁵ imino resonance remained sharp at 55 °C but broadened at 65 °C. In contrast, for the 3′-neighbor A⁸·T¹⁷ base pair, the T¹⁷ imino resonance was severely broadened at 55 °C. Structural refinement using NOE distance restraints obtained from isotope-edited ¹⁵N NOESY-HSQC data indicated that the (<i>R</i>)-γ-hydroxytrimethylene linkage maintained the C⁶·Y¹⁹ and X⁷·C¹⁸ base pairs with minimal structural perturbations. The (<i>R</i>)-γ-hydroxytrimethylene linkage was located in the minor groove. The X⁷ <i>N</i>² and Y¹⁹ <i>N</i>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^α of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C²⁰ <i>O</i>² located in the 5′-neighboring base pair G⁵·C²⁰. 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 (<i>R</i>) configuration of the cross-link