Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae.
The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy-G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA. The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine. In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8-oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine. In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil. The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively. The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction. Furthermore, cleavage of 8-OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4). The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay. In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex. The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated. The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine. On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1.
Girard
P M
PM
Département de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, Radiobiologie Moléculaire et Cellulaire, Fontenay aux Roses, France.
D'Ham
C
C
Cadet
J
J
Boiteux
S
S
D044503:5-Methylcytosine; D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D009841:Oligonucleotides; D011687:Purines; D011725:Pyridines; C002992:8-hydroxyadenine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D000225:Adenine