Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma loVo and Escherichia coli cell extracts

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 laxZα DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZα gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 × 10^-4 in U251 extracts and 5.4 × 10^-4 in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T and G > T to A ≫ T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 × 10^-4. A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.