Sequence specificity of DNA repair by Escherichia coli Fpg protein

The sequence specificity of guanine methylation in DNA by N-methyl-N-nitrosourea and the subsequent repair of ring-opened N7-methylguanine was studied using oligonucleotides of defined sequence. It was found that the methylation of TAGGGGCCCCTA was less than 2-fold greater than that occurring in TAGAGATCTCTA or TATGTGCACATA and 6-fold greater than in TACGCGCGCGTA. This is consistent with the concept that guanine methylation is least when the 5' preceding base is a pyrimidine and greatest when the 5' base is another guanine. These dodecamers were used to study repair by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) after the 7-methyl-guanine present in them was converted to the ring-opened form by alkaline treatment. The repair of ring-opened 7-methylguanine was much faster in self-complementary double-stranded 12mer substrates and was twice as rapid at 37 degrees C in TAGGGGCCCCTA compared with TACGCGCGCGTA. However, at 15 degrees C, the relative rates were reversed since TACGCGCGCGTA was repaired at the same rate as at 37 degrees C, whereas the repair of TAGGGGCCCCTA was much slower at 15 degrees C. The repair of TAGGGGCCCCTA at 37 degrees C was also much faster than the repair of TAGAGATCTCTA and was slightly more rapid than repair of TATGTGCACATA. Ligation of the dodecamer substrates to form 24mers or 36mers slightly reduced the initial rates of repair but did not abolish these differences. These results indicate that under physiological conditions, the Fpg protein is more active against adducts in guanine-rich regions and such regions may be the most likely sites of adduct formation at the N7-position of guanine which can then give rise to derivatives attacked by this enzyme.