The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadu, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA. The repair of damaged DNA is essential for life. The deamination of deoxycytidine to deoxyuridine occurs spontaneously and is also subject to catalysis by UV and bisulphite. It generates a promutagenic U:G mismatch which, if not repaired, leads to a C-T transition mutation in the next round of DNA synthesis.12 The enzyme Uracil-DNA Glycosylase (UDG) identifies the RNA-base uracil in a DNA duplex and hydrolyses the glycosylic linkage between C-1' of the deoxyribose and N-l of uracil with a discrimination of not less than lo7 against dT and dC re~idues.3.~,~ This creates Dedicated to the memory of Sasha Krayevsky, good friend and fine colleague.

Glycosidic Bond Cleavage in DNA Nucleosides: Effect of Nucleobase Damage and Activation on the Mechanism and Barrier

“…This finding is largely consistent with the proposed mechanisms for DNA glycosylases that target pyrimidine derivatives. Specifically, U has been proposed to be removed by hUNG2 in humans or UDG in bacteria through an S N 1 pathway, 5,30,77,78 which correlates with our predicted preferred inherent chemistry (Table 1). Furthermore, a dissociative mechanism has been shown to be favored for TDG-mediated excision of mismatched thymine.…”

Synthesis and Application of 4′-C-Alkylated Uridines as Probes for Uracil-DNA Glycosylase

“…The used derivatives comprise carbocyclic and C-nucleosides, 4¢-thio or 2¢-fluoro-2¢-deoxyuridines and hydrophilic nucleoside surrogates with no hydrogen bonding capacity. [3][4][5][6][7] As shown by structural studies the enzyme UDG flips the RNA base uracil out of the DNA base stack in the active site. 3,8 This process is accompanied with large conformational changes of the enzyme and the DNA substrate.…”