The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis 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. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952-963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.