In humans, uracil appears in DNA at the rate of several hundred bases per c
ell each day as a result of misincorporation of deoxyuridine (dU) or deamin
ation of cytosine. Four enzymes that catalyse the hydrolysis of the glycosy
lic bond of dU in DNA to yield an apyridiminic site as the first step in ba
se excision repair have been identified in the human genome(1). The most ef
ficient and well characterized of these uracil-DNA glycosylases is UDG (als
o known as UNG and present in almost all known organisms)(2), which excises
U from single- or double-stranded DNA and is associated with DNA replicati
on forks(3). We used a hybrid quantum-mechanical/molecular-mechanical (QM/M
M) approach(4) to determine the mechanism of catalysis by UDG. In contrast
to the concerted associative mechanism proposed initially (5-10), we show h
ere that the reaction proceeds in a stepwise dissociative manner(11,12). Cl
eavage of the glycosylic bond yields an intermediate comprising an oxocarbe
nium cation and a uracilate anion. Subsequent attack by a water molecule an
d transfer of a proton to D145 result in the products. Surprisingly, the pr
imary contribution to lowering the activation energy comes from the substra
te, rather than from the enzyme. This 'autocatalysis' derives from the buri
al and positioning of four phosphate groups that stabilize the rate-determi
ning transition state. The importance of these phosphates explains the resi
dual activity observed for mutants that lack key residues(6-9). A correspon
ding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1,
which belong to the same structural superfamily as UDG(13,14).