title: On the formation and properties of interstrand DNA-DNA cross-links forged by reaction of an abasic site with the opposing guanine residue of 5'-CAp sequences in duplex DNA.
abstract: 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'-CAp sequence under conditions of reductive amination (NaCNBH(3), 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-MS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N(2)-amino group of the opposing guanine residue in 5'-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH(3)/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N(2)-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 (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH(3).
authors.affiliation: Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Johnson
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Price
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Mostafa I
authors.initials: MI
authors.lastname: Fekry
authors.forename: Sanjay
authors.initials: S
authors.lastname: Dutta
authors.forename: Derrick R
authors.initials: DR
authors.lastname: Seiner
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
authors.forename: Kent S
authors.initials: KS
authors.lastname: Gates
mesh_terms: D020121:5' Untranslated Regions; D000447:Aldehydes / Q000737:chemistry; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D053719:Tandem Mass Spectrometry
chemical_list: D020121:5' Untranslated Regions; D000447:Aldehydes; D003432:Cross-Linking Reagents; D006147:Guanine; D004247:DNA