Structural basis for uracil DNA glycosylase interaction with uracil: NMR study

Abstract

Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The ¹H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T¹² and T¹⁵ nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T¹⁴ stacks upon both T¹² and T¹⁵. Though U13 partially stacks upon T¹⁴, no stacking interaction is observed between U13 and T¹². All the individual nucleotide bases belonging to the stem and T¹² and T¹⁵ of the loop adopt 'anti' conformation with respect to their sugar moiety, while the U13 and T¹⁴ of the loop are in 'syn' conformation. The turning phosphate in the loop is located between T¹³ and T¹⁴. This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. Theε of T₁₂ and α , β , γ , ε and ζ of U₁₃ and γ of T₁₄, which partially influence the local conformation of U₁₃ in U2-hairpin are all locked in 'trans' conformation. Such stretched out backbone conformation in the vicinity of U₁₃ could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.