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NMR Solution Structures of Clustered Abasic Site Lesions in DNA: Structural Differences between 3′-Staggered (−3) and 5′-Staggered (+3) Bistranded Lesions

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journal contribution
posted on 19.10.2010, 00:00 by Raphael D. Hazel, Carlos de los Santos
Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relatively low number of clustered lesions may be lethal to cells when compared to a larger number of single lesions. Enzyme cleavage experiments suggest that the orientation of bistranded lesions causes differential recognition and removal of these lesions. Like that of a previous study of bistranded abasic site lesion [Hazel, R. D., Tian, K., and de los Santos, C. (2008) Biochemistry 47, 11909−11919], the aim of this investigation was to determine the structures of two DNA duplexes each containing two synthetic apurinic/apyrimidinic (AP) residues, positioned on opposite strands and separated by two base pairs. In the first duplex, the AP residues are staggered in the 3′ orientation [−3 duplex, (AP)2−3 duplex], while in the second duplex, the AP residues are staggered in the 5′ orientation [+3 duplex, (AP)2+3 duplex]. NOESY spectra recorded in 100 and 10% D2O buffer solutions allowed the assignment of the nonexchangeable and exchangeable protons, respectively, for each duplex. Cross-peak connectivity in the nonexchangeable proton spectra indicates that the duplex is a regular right-handed helix with the AP residues and orphan bases located inside the duplexes. The exchangeable proton spectra establish the formation of Watson−Crick G·C alignment for the two base pairs between the lesion sites in both duplexes. Distance-restrained molecular dynamics simulation confirmed the intrahelical orientations of the AP residues. The proximity of the AP residues across the minor groove of the −3 duplex and across the major groove in the +3 duplex is similar to their locations in the case of −1 and +1 clusters. This difference in structure may be a key factor in the differential recognition of bistranded AP lesions by human AP endonuclease.