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The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence.

ABSTRACT: The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.

SUBMITTER: Huang H

PROVIDER: S-EPMC2646759 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence.

Huang Hai H Wang Hao H Qi Nan N Lloyd R Stephen RS Rizzo Carmelo J CJ Stone Michael P MP

Biochemistry 20081011 44

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basi ...[more]

PMID: 18847226

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Project description:The solution structures of 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' interstrand DNA cross-links in the 5'-CpG-3' sequence were determined by NMR spectroscopy. These were utilized as chemically stable surrogates for the corresponding carbinolamine interstrand cross-links arising from the crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts. The results provide an explanation for the observation that interstrand cross-link formation in the 5'-CpG-3' sequence by the R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts is dependent upon stereochemistry, favoring the R-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adduct [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61]. Molecular dynamics calculations, restrained by NOE-based distances and empirical restraints, revealed that both the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-links were located in the minor groove and retained Watson-Crick hydrogen bonds at the tandem cross-linked C.G base pairs. However, for the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the center of the minor groove, whereas for the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the 3' direction, showing steric interference with the DNA helix. The 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link exhibited a lower thermal stability as evidenced by NMR spectroscopy as a function of temperature. The two cross-links also exhibited apparent differences in the conformation of the interstrand three-carbon cross-link, which may also contribute to the lower apparent thermodynamic stability of the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link.

Project description:The crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts were monitored in single-stranded and duplex oligodeoxynucleotides using NMR spectroscopy. In both instances, the cis and trans diastereomers of the alpha-CH3 and gamma-OH groups underwent slow exchange, with the trans diastereomers being favored. In single-stranded oligodeoxynucleotides, the aldehyde intermediates were not detected spectroscopically, but their presence was revealed through the formation of N-terminal conjugates with the tetrapeptide KWKK. When annealed into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCYCTAGC)-3' containing the 5'-CpG-3' sequence context (X = R- or S-alpha-CH3-gamma-13C-OH-PdG; Y = 15N2-dG) at pH 7, partial opening of the R- or S-alpha-CH3-gamma-13C-OH-PdG adducts to the corresponding N2-(3-oxo-1-methyl-propyl)-dG aldehydes was observed at temperatures below the T(m) of the duplexes. These aldehydes equilibrated with their geminal diol hydrates; higher temperatures favored the aldehydes. When annealed opposite T, the S-alpha-CH3-gamma-13C-OH-PdG adduct was stable. At 37 degrees C, an interstrand DNA cross-link was observed spectroscopically only for the R-alpha-CH3-gamma-OH-PdG adduct. Molecular modeling predicted that the interstrand cross-link formed by the R-alpha-CH3-gamma-OH-PdG adduct introduced less disruption into the duplex structure than did the cross-link arising from the S-alpha-CH3-gamma-OH-PdG adduct, due to differing orientations of the R- and S-CH3 groups. Modeling also predicted that the alpha-methyl group of the aldehyde arising from the R-alpha-CH3-gamma-OH-PdG adduct is oriented in the 3'-direction in the minor groove, facilitating cross-linking. In contrast, the alpha-methyl group of the aldehyde arising from the S-alpha-CH3-gamma-OH-PdG adduct is oriented in the 5'-direction within the minor groove, potentially hindering cross-linking. NMR revealed that for the R-alpha-CH3-gamma-OH-PdG adduct, the carbinolamine form of the cross-link was favored in duplex DNA with the imine (Schiff base) form of the cross-link remaining below the level of spectroscopic detection. Molecular modeling predicted that the carbinolamine linkage maintained Watson-Crick hydrogen bonding at both of the tandem C.G base pairs. Dehydration of the carbinolamine cross-link to an imine, or cyclization of the latter to form a pyrimidopurinone cross-link, required disruption of Watson-Crick hydrogen bonding at one or both of the cross-linked base pairs.

Project description: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.

Project description:DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O6-dG-derived ICLs. Although less abundant, O6-alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O6-2'-deoxyguanosine-butylene-O6-2'-deoxyguanosine (O6-dG-C4-O6-dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O6-dG-C4-O6-dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol ? is completely inhibited by O6-dG-C4-O6-dG ICLs. Moderate bypass activities are observed for human pol ? and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol ? exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol ? in O6-dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol ?. Together, this study provides the first set of biochemical evidence for the mutagenicity of O6-dG-derived ICLs.

Project description:Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-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(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'·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 adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) 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 Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were 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, 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 C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.

Dataset Information

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence.

Publications

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence.

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