Uracil-DNA glycosylase acts by substrate autocatalysis

Citation
Ar. Dinner et al., Uracil-DNA glycosylase acts by substrate autocatalysis, NATURE, 413(6857), 2001, pp. 752-755
Citations number
30
Categorie Soggetti
Multidisciplinary,Multidisciplinary,Multidisciplinary
Journal title
NATURE
ISSN journal
00280836 → ACNP
Volume
413
Issue
6857
Year of publication
2001
Pages
752 - 755
Database
ISI
SICI code
0028-0836(20011018)413:6857<752:UGABSA>2.0.ZU;2-6
Abstract
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).