Figure 1.

(Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.