Base-flipping mutations of uracil DNA glycosylase: substrate rescue using a pyrene nucleotide wedge |
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Authors: | Jiang Yu Lin Stivers James T Song Fenhong |
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Affiliation: | Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institutes, and The National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA. |
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Abstract: | We recently introduced a new substrate rescue tool for investigating enzymatic base flipping by uracil DNA glycosylase (UDG) in which a bulky pyrene nucleotide wedge (Y) was placed opposite a uracil in duplex DNA (i.e., a U/Y pair), thereby preorganizing the target base in an extrahelical conformation [Jiang, Y. L., et al. (2001) J. Biol. Chem. 276, 42347-54]. The pyrene wedge completely rescued the large catalytic defects resulting from removal of the natural Leu191 wedge, presumably mimicking the pushing and plugging function of this group. Here we employ the pyrene rescue method in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism (see the preceding paper in this issue). We find that a U/Y base pair increases the apparent second-order rate constant for damaged site recognition by L191G pushing mutation by 45-fold as compared to a U/A pair, thereby fully rescuing the kinetic effects of the mutation. Remarkably, the U/Y pair also allows L191G to proceed through the conformational docking step that is severely comprised with the normal U/A substrate, and allows the active site of UDG to clamp around the extrahelical base. Thus, pyrene also fulfills the plugging role of the Leu191 side chain. Preorganization of uracil in an extrahelical conformation by pyrene allows diffusion-controlled damage recognition by all of these base-flipping mutants, and allows the UDG conformational change to proceed as rapidly as the rate of uracil flipping with the natural U/A base pair. Thus, the pyrene wedge substrate allows UDG to recognize uracil by a lock-and-key mechanism, rather than the natural induced-fit mechanism. Unnatural pyrene base pairs may provide a general strategy to promote site-specific targeting of other enzymes that recognize extrahelical bases. |
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