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

Russell J. Sanderson, Samuel E. Bennett, Jung Suk Sung, Dale W. Mosbaugh

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

10 Scopus citations

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 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.

Original languageEnglish
Title of host publicationBase Excesion Repair
PublisherAcademic Press Inc.
Pages165-188
Number of pages24
ISBN (Print)0125400683, 9780125400688
DOIs
StatePublished - 2001

Publication series

NameProgress in Nucleic Acid Research and Molecular Biology
Volume68
ISSN (Print)0079-6603

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