On the Formation and Properties of Interstrand DNA鈥揇NA Cross-Links Forged by Reaction of an Abasic Site with the Opposing Guanine Residue of 5鈥?CAp Sequences in Duplex DNA

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• 作者：Kevin M. Johnson ; Nathan E. Price ; Jin Wang ; Mostafa I. Fekry ; Sanjay Dutta ; Derrick R. Seiner ; Yinsheng Wang ; Kent S. Gates
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：January 23, 2013
• 年：2013
• 卷：135
• 期：3
• 页码：1015-1025
• 全文大小：492K
• 年卷期：v.135,no.3(January 23, 2013)
• ISSN：1520-5126

文摘

We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5鈥?CAp sequence under conditions of reductive amination (NaCNBHb>3b>, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions, and the cross-link, once formed, was stable to a variety of workup conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LC鈥揗S/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N²-amino group of the opposing guanine residue in 5鈥?CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (<b>7b>鈥?b>10b>) that were irreversibly converted under conditions of reductive amination (NaCNBHb>3b>/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N²-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15鈥?0%) of a cross-linked duplex at neutral pH, in the absence of NaCNBHb>3b>.