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 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.
Sanderson
R J
RJ
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Bennett
S E
SE
Sung
J S
JS
Mosbaugh
D W
DW
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016590:Aphidicolin / Q000494:pharmacology; D017104:Bacteriophage M13 / Q000235:genetics; D002457:Cell Extracts; D002474:Cell-Free System; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon / Q000187:drug effects; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014498:Uracil / Q000502:physiology*; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000502:physiology*
D002457:Cell Extracts; D004269:DNA, Bacterial; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D009363:Neoplasm Proteins; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D016590:Aphidicolin; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase