Simple, High‐Yield Syntheses of DNA Duplexes Containing Interstrand DNA‐DNA Cross‐Links Between an N⁴‐Aminocytidine Residue and an Abasic Site

Abstract

The protocol describes the preparation and purification of interstrand DNA-DNA cross-links derived from the reaction of an N4-aminocytidine residue with an abasic site in duplex DNA. The procedures employ inexpensive, commercially-available chemicals and enzymes to carry out post-synthetic modification of commercially-available oligodeoxynucleotides. The yield of cross-linked duplex is typically better than 90%. If purification is required, the cross-linked duplex can be readily separated from single-stranded DNA starting materials by denaturing gel electrophoresis. The resulting covalent hydrazone-based cross-links are stable under physiologically-relevant conditions and may be useful for biophysical studies, structural analyses, DNA repair studies, and materials science applications.

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

“…We also observed a “late-forming” cross-link band that became visible after 12 h and predominant after 48 h [4% yield (Figure )]. It seemed unlikely that this could be a drug-linked cross-link because the chloroethyl groups of HN2 in both DNA monoadducts [ 1 (Scheme )] and free HN2 hydrolyze rapidly ( t 1/2 = 1–30 min). ,− On the other hand, in light of our recent studies, − it was reasonable to consider that a buildup of Ap sites resulting from depurination of HN2 adducts ( 1–3 ) might permit the generation of Ap-derived cross-links. In the following sections, we provide evidence that the late-forming cross-link band seen in Figure A is a dA-Ap cross-link resulting from alkylation-induced generation of an Ap site at position 2 of duplex A (Figure ).…”

“…Toward this end, we conducted a series of experiments in which an authentic Ap site was introduced at position 1, 2, 6, or 8 in duplex A . The 5′- 32 P-labeled Ap-containing duplexes B–E were prepared as described in our previous work − by the action of uracil DNA glycosylase (UDG) on the corresponding 2′-deoxyuridine-containing oligodeoxynucleotide duplexes. , The formation of slowly migrating, Ap-derived cross-link bands was easily detected in each of the Ap-containing duplexes B–E (Figure ). The equilibrium yields of the cross-link generated in these duplexes varied substantially, with values of 5, 30, 14, and 2%, for duplexes B–E , respectively (Figure S1).…”

“… ,,, We recently showed that Ap sites can forge DNA–DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases such as adenine and guanine on the opposing strand of the DNA duplex (Scheme ). − The cross-linking reactions considered here, involving “true” Ap sites, are distinct from those involving oxidized abasic sites. , Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine (HN2) give rise to interstrand cross-links of a type not previously associated with this drug. We present gel electrophoretic data that are consistent with an early evolution of the expected drug-bridged cross-links 3 followed by the appearance of Ap-derived cross-links.…”

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

“…Nicked DNA duplexes containing the 3’ddR5p end group at the break were generated by reaction of the endogenous polyamine spermine ( 60 ) with the corresponding Ap duplexes ( 43 , 45 , 61 ). The 5’- 32 P-labeled, 35 base pair Ap-containing duplex A (Figure 1 ) was prepared from the corresponding 2’-deoxyuridine-containing duplex by treatment with uracil DNA glycosylase (UDG) as described previously ( 53 – 58 , 62 – 64 ). To confirm successful generation of the Ap site in duplex A, denaturing polyacrylamide gel electrophoretic analysis was used to show that treatment with piperidine (1 M, 95°C, 25 min) cleanly induced strand cleavage to give the expected ( 1 , 65 ) fast-migrating product with a 3’-phosphoryl terminus.…”

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

“…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. The propensity for this aldehyde to be attacked by nucleophilic amine residues on the nucleobases of DNA, leading to the formation of interstrand cross-links (ICLs) (Scheme ), is of interest. − …”