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The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.

ortho-Quinone methides (ortho-QM) and para-quinone methides are generated by xenobiotic metabolism of numerous compounds including environmental toxins and therapeutic agents. These intermediates are highly electrophilic and have the potential to alkylate DNA. Assessing their genotoxicity can be difficult when all or some of their resulting adducts form reversibly. Stable adducts are most easily detected but are not necessarily the most prevalent products formed initially as DNA repair commences. Selective oxidation of ortho-QM-DNA adducts by bis[(trifluoroacetoxy)iodo]benzene (BTI) rapidly quenches their reversibility to prevent QM regeneration and allows for observation of the kinetic products. The resulting derivatives persist through standard enzymatic digestion, chromatography, and mass spectral analysis. The structural standards required for this approach have been synthesized and confirmed by two-dimensional NMR spectroscopy. The adducts of dA N(6), dG N1, dG N(2), and guanine N7 are converted to the expected para-quinol derivatives within 5 min after addition of BTI under aqueous conditions (pH 7). Concurrently, the adduct of dA N1 forms a spiro derivative comparable to that characterized previously after oxidation of the corresponding dC N3 adduct. By application of this oxidative quenching strategy, the dC N3 and dA N1 adducts have been identified as the dominant products formed by both single- and double-stranded DNA under initial conditions. As expected, however, these labile adducts dissipate within 24 h if not quenched with BTI. Still, the products favored by kinetics are responsible for inducing the first response to ortho-QM exposure in cells, and hence, they are also key to establishing the relationship between biological activity and molecular structure.

Highly electrophilic quinone methide (QM) intermediates often express a surprising selectivity for weak nucleophiles of DNA even when proximity effects do not guide reaction. On the basis of model studies with an unsubstituted ortho-QM, these observations can now be explained by the reversibility of QM alkylation and the time-dependent shift from kinetic to thermodynamic products. The persistent and most commonly identified QM adducts represent thermodynamic products that typically form in low yield by irreversible reaction with weak nucleophiles such as the N1 and N2 of dG and the N6 of dA under neutral conditions. In contrast, strong nucleophiles such as the N1 of dA and the N3 of dC generate relatively high yields of their QM adducts. However, these products dissipate over time as the QM is repeatedly regenerated and repartitioned over the available nucleophiles. The adduct formed by the N7 of dG undergoes a similar release of QM as well as deglycosylation at comparable rates. The kinetic products of QM alkylation serve as a reservoir for QM regeneration and transfer that are likely to prolong the cellular activity of an otherwise highly transient intermediate.

Quinone methides and related intermediates have been implicated in a range of beneficial and detrimental processes in biology and effectively alkylate a variety of cellular components despite the ubiquitous presence of water. As a prerequisite to understanding the origins of their specificity, the major products generated by DNA and its components with an unsubstituted ortho quinone methide under aqueous conditions were recently characterized [Pande, P., Shearer, J., Yang, J., Greenberg, W. A., and Rokita, S. E. (1999) J. Am. Chem. Soc. 121, 6773-6779]. Investigations currently focus on the complete range of derivatives formed by deoxyguanosine (dG) and guanine residues in duplex DNA through product isolation and structure determination using reversed-phase chromatography and a range of one and two-dimensional NMR techniques. Previous construction of a synthetic standard for dG alkylation is now shown to have yielded the N1-linked adduct rather than the N(2)-linked adduct. This later adduct has also now been characterized and confirmed to be the major product of reaction between the quinone methide and both duplex DNA and dG under neutral conditions. An N7 adduct of guanine has additionally been identified under these conditions and appears to result from spontaneous deglycosylation of the corresponding N7 adduct of dG. A combination of steric and electronic properties of duplex DNA likely contribute to the enhanced selectivity of the quinone methide for its guanine N(2) position (7.8:3.2:1.0 for adducts of N(2):N7:N1) relative to that of dG (4.7:3.5:1.0 for adducts of N(2):N7:N1).

A model protein, ribonuclease A (bovine pancreas), was examined for its ability to coordinate Ni2+ and promote selective oxidation. In the presence of a peracid such as monopersulfate, HSO5-, nickel induced the monomeric RNase A to form dimers, trimers, tetramers, and higher oligomers without producing fragmentation of the polypeptide backbone. Co2+ and to a lesser extent Cu2+ exhibited similar activity. The nickel-dependent reaction appeared to result from a specific association between the protein and Ni2+ that allowed for transient and in situ oxidation of the bound nickel to yield intermolecular tyrosine-tyrosine cross-links. Macrocylic nickel complexes that had previously been shown to promote guanine oxidation were unable to mimic the activity of the free metal salt. Amino acid analysis of the protein dimer confirmed the expected consumption of one tyrosine per polypeptide and formation of dityrosine. The presence of excess tyrosine efficiently inhibited formation of the protein dimer and produced instead a ribonuclease-tyrosine cross-link. In contrast, high concentrations of the hydroxyl radical quenching agent mannitol only partially inhibited ribonuclease dimerization. The polypeptide-mediated activation of nickel and its subsequent reactivity mimic a process that could contribute to the adverse effects of nickel in vivo.