G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by uracil DNA-glycosylase (UDG), which excises uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both uracil and thymine, but only from mispairs with guanine. Crystal structures of the mismatch-specific uracil DNA-glycosylase (MUG) from E. coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity. Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.

Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer-tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.

Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487-493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87-92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single-and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single-or doublestranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases.

Base-excision of a self-complementary oligonucleotide with central G:T mismatches by the G:T/U-specific mismatch DNA glycosylase (MUG), generates an unusual DNA structure which is remarkably similar in conformation to an interstrand DNA adduct of the anti-tumor drug cis-diamminedichloroplatinum. The abasic sugars generated by excision of the mismatched thymines are extruded from the double-helix, and the 'widowed' deoxyguanosines rotate so that their N7 and O6 groups protrude into the minor groove of the duplex and restack in an interleaved intercalative geometry, generating a kink in the helix axis.

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.