Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA

Abstract

The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg 2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH 3 CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES

“…The glycosylase activity of NEIL3 can unhook dA-AP or psoralen ICLs ahead of the stalled CMG helicase at the leading edge of the stalled fork . The dA-AP ICL and perhaps other AP-derived ICLs − may be important endogenous substrates for this DNA repair pathway . It is interesting to consider whether there are other substrates for the NEIL3-dependent prereplication repair pathway.…”

“…AP sites are generated unavoidably in cellular DNA by spontaneous and enzymatic hydrolysis of the glycosidic bonds that connect nucleobases to the phosphodiester backbone. − AP sites in DNA exist as an equilibrium mixture of the ring-closed hemiacetal and the ring-opened aldehyde (shown in Figure A) . In duplex DNA, AP sites can generate ICLs via reactions of the ring-opened AP-aldehyde with exocyclic amino groups of adenine, guanine, and cytosine residues located on the opposing strand (Figure B). − The process involves reversible formation of an imine, followed by ring-closure to generate a stable aminoglycoside cross-link (Figure A). Formation of the dA-AP ICL does not introduce large distortions into the DNA duplex (Figure C) suggesting that the lesion might be only be recognized and repaired by replication-dependent pathways. , When considering potential biological effects of the dA-AP ICL, it is significant that, despite the reversible nature of imine formation, the cross-link is able to block a strand-displacing polymerase and the replicative helicase. , As an aside, AP-derived cross-linking reactions have been exploited in the development of simple biochemical methods for the high-yield preparation of DNA duplexes containing site-specific, structurally defined ICLs − and as a means for single-molecule detection of disease-relevant DNA sequence polymorphisms. − …”

Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions

“…Importantly, the AP-lyase activity is not limited to these well-studied enzymes. A body of work by the Greenberg and Gates laboratories has shown that free amino groups in DNA-binding proteins and nucleobases can catalyze similar reactions. − For example, Greenberg and associates have demonstrated that Lys residues of histone tails can promote strand scission at AP sites in reconstituted nucleosome particles. , Gates and associates have shown that in vitro amino groups of the nucleoside 3′ or 5′ of the nucleoside opposing the AP lesion can catalyze similar reactions in duplex DNA substrates with a variety of DNA sequence contexts and that biological amines, such as spermine, can accelerate the reaction. − …”

Ultrafast Reaction of the Drug Hydralazine with Apurinic/Apyrimidinic Sites in DNA Gives Rise to a Stable Triazolo[3,4-a]phthalazine Adduct

“…The 5′-Cy5-labeled oligonucleotides were HPLC-purified before use. The 5′-labeled dU-containing oligonucleotide was hybridized with the complementary oligonucleotide and treated with the enzyme uracil DNA glycosylase (UDG, final concentration of 200 units/mL) in Tris–HCl buffer (20 mM, pH 8) containing dithiothreitol (DTT, 1 mM) and EDTA (1 mM). − The UDG was removed via phenol–chloroform–isoamyl alcohol extraction, and the AP-containing DNA was ethanol-precipitated. The yield of AP site formation was typically well over 90%, as monitored by NaOH workup (usually 200 mM, 20 min, 37 °C) to induce strand cleavage.…”