Chemical Research in Toxicology ARTICLE Figure 5. Melting studies of the DNA KWKK conjugates by UV vis (254 nm) at pH 7.0 in 100 mM NaCl: —)— ﬁrst experiment; —2— second experiment; —9— third experiment; —b— fourth experiment. of B-DNA structure; in contrast, imine 6 and pyrimidopurinone 7 interstrand linkages perturb DNA base pairing and base stacking.17,44,61 Additionally, the potential for hydrogen bonding between the carbinol and the DNA probably stabilizes the linkages and may account for the reaction stereoselectivity.62 Similarly, carbinolamine linkages have been trapped in the active sites of type I aldolases; their presence as compared to imines has been attributed to their stabilization by hydrogen bonding interactions.63 65 Structures of Carbinolamine DNA KWKK Conjugates. Recently, we reported the structure of the reduced conjugate formed between the KWKK peptide and the γ-OH-PdG adduct.18 Watson Crick hydrogen bonding was conserved at the site of conjugation, the peptide was oriented in the minor groove, and the tryptophan indolyl group did not intercalate into the DNA. At the site of conjugation, the guanine N2 amine nitrogen and the amine nitrogen of the N-terminal lysine were in the trans-configuration with respect to the CR or Cγ of the trimethylene tether, respectively. The reduced DNA KWKK conjugate thermally stabilized the DNA. The reﬁned structure of the reduced cross-link provided a basis for modeling of the native diastereomeric carbinolamine conjugates. The minimal chemical shift changes noted for the DNA protons and their similar values as compared to those of the reduced conjugate suggest that the diastereomeric carbinolamine DNA KWKK conjugates have structures similar to that of the reduced DNA KWKK conjugate. Molecular modeling suggests that the major stereoisomer of the carbinolamine DNA KWKK conjugate places the peptide in the minor groove, similar to that observed for the reduced analogue (Figure 6). Base pair X7 3 C18 conserves Watson Crick hydrogen bonding. However, the major carbinolamine conjugate exhibits a weak X7 H8 f C6 H10 NOE correlation, while the C6 3 G19 and X7 3 C18 base pairs exhibit small perturbations. 1,N2-propano-20-deoxyguanosine (PdG) has been used to model the 1,N2-dG adducts, and the PdG 3 C base pair has been well-studied.43,66 In acidic solution, PdG adopts the syn con- formation about the glycosyl bond to enable protonated Hoog- steen hydrogen bonding with the complementary cytosine. The evident Watson Crick hydrogen bonding of X7 3 C18 of the major DNA KWKK conjugate was consistent with carbinola- mine 7 being the predominant species. Figure 6. Expanded model structures of the diastereomeric carbinola- mine N2-dG:N-Lys DNA KWKK conjugates viewed from the minor groove: (A) (R)-conﬁguration, (B) (S)-conﬁguration. Blue, red, green, and pink sticks represent oligonucleotide, peptide, trimethylene, and the hydroxyl group, respectively. Table 1. Hydrogen Bond Occupancies of the Carbinol Hydroxyl Group by the Acceptors during MD Simulations hydrogen bond occupation (%)a receptor R-conﬁguration S-conﬁguration X7 N3 0.2 0.0 A8 O40 5.2 0.0 G19 O40 0.0 29.6 K25 CdO 0.0 4.2 water 8.8 2.2 a Criteria for the hydrogen bond formation: distance <3.5 Å and angle >120 . Nucleotide Excision Repair of DNA KWKK Conjugates. These lesions are anticipated to be substrates for the NER pathway. The kinetics of incision of UvrABC have been measured using either KWKK- or KFHEKHHSHRGY-peptide DNA conjugates or the T4-pdg DPC, conjugated to duplex DNA via γ-OH-PdG chemistry.14,67 In all instances, all UvrABC proteins were necessary to catalyze the incision reactions. Incision of DNA containing the KFHEKHHSHRGY-peptide conjugate at either the abasic or γ-OH-PdG site is significantly greater than the incision of DNA containing a fluorescein-conjugated dT.68 The KFHEKHHSHRGY- peptide attached via an abasic site is incised 3-times more efficiently than that when conjugated via γ-OH-PdG. Genotoxicity of DNA KWKK Conjugates. Modified oligo- deoxynucleotides generated by conjugating KWKK to γ-OH-PdG have been inserted into a single-stranded pMS2 shuttle vector replicated in COS-7 cells.16 Replication bypass of the γ-OH-PdG- mediated DNA KWKK conjugate results in mutations at the site of modification at an overall frequency of ∼8.4%. The γ-OH-PdG- mediated DNA KWKK conjugate yields predominantly single base substitutions, with a prevalence of G to T transversions. The ability of the DNA peptide conjugates generated by linking KWKK to γ-OH-PdG to be bypassed by both human and E. coli polymerases in vitro have also been investigated.69 Human DNA polymerase κ catalyzes eﬃcient, error-free bypass 1130 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133