Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiest er backbone of damaged DNA using the hydroxyl side chains of a conserved tr io of serine residues, resulting in flipping of the deoxyuridine from the D NA helix into the enzyme active site. We have investigated the energetic ro le of these serine-phosphodiester interactions using the complementary appr oaches of crystallography, directed mutagenesis, and stereospecific phospho rothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAA p-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, an d that the enzyme has undergone the same specific clamping motion that brin gs key active site groups into position as previously observed in the struc tures of human UDG bound to large duplex DNA substrates. From this structur e, it may be concluded that glycosidic bond cleavage and the induced fit co nformational change in UDG can occur without the 5' pinching interaction. T he S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fo ld damaging effects on k(cat)/K-m, respectively, while the S88A/S189A doubl e mutant exhibits an 8200-fold damaging effect. A free energy analysis of t he combined effects of nonbridging phosphorothioate substitution and mutati on at these positions reveals the presence of a modest amount of strain ene rgy between the compressed 5' and 3' phosphodiester groups flanking the bou nd uridine. Overall, these results indicate a role for these serine-phospho diester interactions in uracil flipping and preorganization of the sugar ri ng into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Scf. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serin e pinching plays a major role in the 10(12)-fold rate enhancement brought a bout by UDG.