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J. Biol. Chem., Vol. 276, Issue 45, 42588-42600, November 9, 2001

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Fidelity of Uracil-initiated Base Excision DNA Repair in DNA Polymerase -Proficient and -Deficient Mouse Embryonic Fibroblast Cell Extracts^*

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

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

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase  (+/+) and (/) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U·A, U·G, and U·T targets contained within the lacZ gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase  (+/+) and (/) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (~88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting ~20% of the repair events. Addition of p21 peptide or protein to DNA polymerase  (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by ~2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U·T) DNA was determined to be 5.7-7.2 × 10⁴ when using DNA polymerase  (+/+) and (/) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U·G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U·G target and frequently involved a 1-base deletion or incorporation of dTMP.

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