Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

Abstract

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We Common forms of DNA damage, for instance those induced spontaneously by reactive metabolites produced inside the cells, are handled by the base excision repair pathway (1, 2). Different types of oxidized and alkylated base residues are recognized by different DNA glycosylases that release the bases in a free form and leave abasic sites in the DNA. Abasic sites are subsequently removed by the combined action of an apuriniclapyrimidinic (AP)-endonuclease and a 5'-phosphodiesterase or, alternatively, by an AP-lyase and a 3'-phosphodiesterase. Base excision repair is completed by repair synthesis to fill the one nucleotide gap and ligation to restore the continuity of the DNA strand. Other protein factors are probably also involved in the base excision repair pathway in vivo (2).The DNA glycosylases are responsible for damage recognition and as such are key enzymes in the repair process. These can be classified into a few different subtypes, mainly based on genetic and biochemical characterization of DNA glycosylases in bacteria (2). Two of these are primarily involved in the removal of oxidative DNA damage, i.e., endonuclease III (EndoIII) and the formamidopyrimidine/8-oxoguanine DNA glycosylase (Fpg), which in a broad sense are responsible for the removal of oxidized pyrimidine and purine damage, respectively (3-6). Both of these enzymes have intrinsic AP-lyase activity, whereas other enzymes involved in the removal of deaminated and alkylated base residues do not (2). Both EndollI and Fpg have been extensively characterized from Escherichia coli and other bacteria; however, attempts to define the functional role of analogous enzymes in eukaryotes have been hampered by difficulties in cloning the corresponding genes. Enzyme activities similar to EndoIllI and Fpg have been purified from yeast (7,8) and mammalian cells (9, 10); however, limited amounts of the enzymes in such species have prevented purification to homogeneity and cloning by the approach of protein sequencing.Since all DNA glycosylases cleave the N-glycosylic bond between the base and the deoxyribose moieties of the nucleotides, one might have expected to find a common sequence domain that would be responsible for the cleavage reaction. However, different DNA glycosylases are largely unrelated in terms of sequence with the exception of EndolIl and MutY, which are of common evolutionary origin (11). Nevertheless, local sequence alignments have identified a subgroup of DNA glycosylases, including EndollI and MutY, that share a characteristic helix-hairpin-helix domain. This domain appears to be involved in determining the specificity of the enzymes for reacting with different types of DNA damage (2, 12). The MutY enzyme removes adenosines from A-8-oxoguanine (8-oxoG) mispairs to prevent mutations arising from the misincorporation o...

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…Ntg1, which excises base-derived lesions from oxidatively damaged DNA, has AP-lyase (previously described as endonuclease) activity (15,45; for review, see reference 26). From its known activity, Ntg1 is expected to produce single-stranded breaks (nicks) rather than double-stranded breaks.…”

“…Ntg1 has DNA repair glycosylase and apurinic/apyrimidinic (AP) lyase activities. It recognizes various oxidatively modified bases and causes singlestranded breakage (nicking) in double-stranded DNA (15,45,55). Thus, the effect of NTG1 disruption on ori5-specific double-stranded cleavage is not readily explainable by the known biochemical activities of the Ntg1 protein.…”

“…The standard reaction mixture (10 l) consisted of 0.2 g total DNA digested with BglII, 70 mM 3-(N-morpholino) propanesulfonic acid (pH 7.5), 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and various concentrations of the Ntg1 or C⌬ntg1 protein. The reaction was carried out at 37°C for 30 min as described elsewhere (15). After removing the proteins from the reaction solution with proteinase K, the digests were analyzed by electrophoresis on a 2% agarose gel.…”

“…No double-stranded cleavage was detected under these conditions. Thus, the Ntg1 protein purified from E. coli has the specific activity described previously (15).…”

DNA glycosylases in the base excision repair of DNA

“…Subsequently, genes or cDNAs encoding homologues of EndoIII have been identified in Bacillus subtilis [184], Haemophilus influenzae [185], Methanococcus jannaschii [186], S. cere isiae (NTG1) [187], S. pombe (Nth-Spo) [188], Candidas elegans [189], rat (EMBL\GenBank accession no. H33255) and human [190,191].…”