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J. Biol. Chem., Vol. 276, Issue 3, 2276-2285, January 19, 2001

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Fidelity of Uracil-initiated Base Excision DNA Repair in Escherichia coli Cell Extracts^*

Jung-Suk Sung, Samuel E. Bennett, and Dale W. Mosbaugh^§¶

From the Departments of  Environmental and Molecular Toxicology and ^§ Biochemistry and Biophysics and the ^¶ Environmental Health Science Center, Oregon State University, Corvallis, Oregon 97331-7301

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZ DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung⁺ dug⁺), ~80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung dug⁺) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung dug⁺), BH157 (ung⁺ dug), and BH158 (ung dug) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be ~5.5 × 10⁴ with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.

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