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

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)/K(m), 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 10(12)-fold rate enhancement brought about by UDG.

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

“…To evaluate whether MS/MS can locate multiple abasic sites in ODNs, we chose T2X5U and CT3X5U (Table 1, X ϭ uracil) as test compounds. We found that UDG only selectively removed the 5'-uracil bases from these ODNs, leaving the 3'-uracil base intact, an observation that is consistent with results in the literature [31][32][33]. When we treated T49X (Table 1, X ϭ uracil), containing two mid-chain uracil bases, using the same reaction conditions as those for T2X5U and CT3X5U and submitted the product to ESI analysis, we found the [ (Table 1).…”

Presteady-state Analysis of a Single Catalytic Turnover by Escherichia coli Uracil-DNA Glycosylase Reveals a “Pinch-Pull-Push” Mechanism

“…Previous reports in the literature have measured isolated segments of the reaction pathway using different substrates (20,32,38,39). It is not possible to test the self-consistency of a global mechanism comprising the sum of segments derived from experiments using different substrates.…”

The Role of Leucine 191 of Escherichia coliUracil DNA Glycosylase in the Formation of a Highly Stable Complex with the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic Substrates

“…Thus, our results point out to the role of Leu 191 at a step utilized by both the single-and double-stranded substrates in common. These observations can be easily rationalized if the interaction between the Ser 88 , Ser 189 , and Ser 192 and the phosphates flanking the target uridine (37)(38)(39), which lead to the compression (the "pinch" step) of the sugar phosphate backbone, was the first event to take place in the substrate recognition. The kinking of the backbone would result in destabilization of the hydrogen bonds of the target uracil with its complement A or G. Thus, much of the discrimination that is seen in utilization of substrates containing uracil in different structural contexts (23,40) should occur at the step of interaction of UDG with the DNA backbone.…”