Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The k _cat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo⁸G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The K _M values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N ⁴- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.