PMID:11554295
| Citation |
Sanderson, RJ, Bennett, SE, Sung, JS and Mosbaugh, DW (2001) Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts. Prog. Nucleic Acid Res. Mol. Biol. 68:165-88 |
|---|---|
| Abstract |
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 lacZ alpha 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 lacZ alpha 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 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to 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 x 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. |
| Links | |
| Keywords |
Adenocarcinoma/genetics; Adenocarcinoma/metabolism; Adenocarcinoma/pathology; Aphidicolin/pharmacology; Bacteriophage M13/genetics; Cell Extracts; Cell-Free System; Colonic Neoplasms/genetics; Colonic Neoplasms/metabolism; Colonic Neoplasms/pathology; DNA Damage; DNA Glycosylases; DNA Repair/drug effects; DNA Repair/physiology; DNA Replication; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA, Neoplasm/genetics; DNA, Neoplasm/metabolism; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Lac Operon/drug effects; Mutation; N-Glycosyl Hydrolases/physiology; Neoplasm Proteins/physiology; Tumor Cells, Cultured/drug effects; Tumor Cells, Cultured/metabolism; Uracil/physiology; Uracil-DNA Glycosidase; Viral Proteins/physiology |
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