Rm. Werner et al., Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase, BIOCHEM, 39(41), 2000, pp. 12585-12594
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.