A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links

Abstract

Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Monoadducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5'-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483-3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the first characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues.

DNA Crosslinkomics: A Tool for the Comprehensive Assessment of Interstrand Crosslinks Using High Resolution Mass Spectrometry

“…In addition to the methylene-bridged crosslinks, this study also observed the dA-AP crosslink in the formaldehyde-treated DNA (Figure C). This class of crosslinks, comprising a 2′-deoxyribonucleoside linked to an apurinic/apyrimidinic site, includes dA-AP and dG-AP and was first identified by the Gates research group ,, in duplex DNA using NMR and low resolution MS. It has been suggested that this class of ICLs has persistent lesions (e.g., half-life: 3–4 days for dA-AP and 22 days for dG-AP at neutral pH) with the potential to block activities associated with DNA processing (e.g., replication).…”

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

“…Interstrand cross-links introduced by bifunctional alkylating agents such as nitrogen mustard anticancer drugs[4–7] prevent strand separation and compromise critical cellular functions of duplex DNA [8, 9]. As a result, DNA cross-links are highly toxic to cells [10, 11].…”

Structure of a Stable Interstrand DNA Cross-Link Involving a β-N-Glycosyl Linkage Between an N6-dA Amino Group and an Abasic Site

“…Abasic (AP) sites in DNA result from spontaneous depurination of canonical and chemically modified nucleobases. − They are also formed by the removal of damaged bases by DNA glycosylases as a part of the base excision repair (BER) pathway . Spontaneous depurination has been estimated to generate 10 4 AP sites per cell per day, with recent studies measuring levels of AP sites in cellular DNA in the range of 1 × 10 –7 /nt. , AP sites, which equilibrate between α and β anomers via the ring-opened aldehyde form of the 2′-deoxyribosyl ring, − are genotoxic, with different bases incorporated opposite the lesion depending on the polymerase and sequence context. , AP-containing duplexes retain a B-DNA conformation. , The presence of an AP site places equilibrium amounts of a ring-opened electrophilic aldehyde in the DNA double helix.…”