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

Abstract

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.

Figure 1

Structures of α,β-unsaturated aldehydes acrolein, crotonaldehyde, and 4-HNE and their cyclic 1,N²-dG adducts.

Scheme 1. 1,N²-dG Cyclic Adducts Arising from Michael Addition of Enals to dG

Scheme 2. Site-Specific Synthesis of Oligodeoxynucleotides Containing 1,N²-dG Adducts of Acrolein, Crotonaldehyde, and 4-HNE by the Postsynthetic Modification Strategy

Figure 2

Model substrates for the γ-HO-PdG adduct in DNA. Top left: PdG (14), a model for the ring-closed form of γ-HO-PdG (2). Top right: PdG forms a Hoogsteen pair with dC in duplex DNA. Bottom left: Reduced γ-HO-PdG adduct (15), a model for the ring-opened N²-(3-oxopropyl)-dG (1). Bottom right: N²-(3-oxopropyl)-dG (1) forms a Watson−Crick pair with dC in duplex DNA.

Scheme 3. Ring-Opening Chemistry of the M₁dG Adduct Opposite dC in Duplex DNA

Scheme 4. Ring-Opening and Cross-Linking Chemistry of 1,N²-Enal-Derived dG Adducts Opposite dC in Duplex DNA

Scheme 5. (6S,8R,11S)- and (6R,8S,11R)-4-HNE-dG Adducts (6 and 7) Form Cyclic Hemiacetals after Initial Ring Opening Opposite dC in Duplex DNA

Figure 3

Oligodeoxynucleotides containing the reduced acrolein-mediated N²-dG:N²-dG interstrand cross-link (23).