Stressing-Out DNA? The Contribution of Serine-Phosphodiester Interactions in Catalysis by Uracil DNA Glycosylase

Published

October 17, 2000

Author(s)

R M. Werner, Y. L. Jiang, R. G. Gordley, J. Jagadeesh, Jane E. Ladner, G Xiao, M Tordova, G L. Gilliland, J T. Stivers

Abstract

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5 -HO-dUAAp-3 (which lacks the 5 phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5 pinching interaction. The S88A, S189A, and S192G pinching mutations exhibit 360-, 80-, and 21-fold damaging effects on k_cat/Km, respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5 and 3 phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S.S. et al, (2000) Proc Natl. Acad. Sci. 94, 5083] there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10¹²-fold rate enhancement brought about by UDG.

Citation

Biochemistry

Volume

39

Issue

41

Pub Type

Journals

Keywords

e.coli uracil DNA glycosylase, nonbridging, site-directed mutagenesis, thio-effects, x-ray crystal structure

Citation

Werner, R. , Jiang, Y. , Gordley, R. , Jagadeesh, J. , Ladner, J. , Xiao, G. , Tordova, M. , Gilliland, G. and Stivers, J. (2000), Stressing-Out DNA? The Contribution of Serine-Phosphodiester Interactions in Catalysis by Uracil DNA Glycosylase, Biochemistry (Accessed October 16, 2024)