Abstract
Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) 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 lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) 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 ( approximately 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 approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 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 x 10(-4) when using DNA polymerase beta (+/+) 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.
Highlights
Uracil residues accumulate in DNA following incorporation of dUMP in place of dTMP during DNA synthesis as well as by spontaneous deamination of existing cytosine residues that form premutagenic U1⁄7G mispairs [1,2,3]
The results showed that one-nucleotide repair patches were the single most prevalent patch size in both POL  (ϩ/ϩ) and POL  (Ϫ/Ϫ) BER reactions, constituting ϳ23 and ϳ18% of total repair patches, respectively
E. coli NR9162 cells were transfected with the form I DNA, and the M13mp2 lacZ␣ DNA-based reversion assay was performed as described under “Experimental Procedures.”
Summary
Materials—PCR primers FP-18-mer, RP-18-mer, and DNA sequencing primer S-21-mer have been described previously [58]. Fractions (1 ml) were collected, and those enriched for p21 protein as determined by 12% SDS-polyacrylamide gel electrophoresis [25] were pooled and extensively dialyzed at 4 °C against AE buffer (2 M urea, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 10% (w/v) glycerol). UracilDNA glycosylase reaction mixtures (100 l) contained 70 mM HepesKOH (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 10 g [uracil-3H]calf thymus DNA (specific activity 150 cpm/pmol), 50 g of MEF POL  (ϩ/ϩ) cell-free extract, and Ugi as indicated. Base Excision DNA Repair Reactions—Standard BER reaction mixtures (100 l) contained 100 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 2 mM ATP, 0.5 mM -NAD, 20 M each of dATP, dTTP, dGTP, and dCTP, 5 mM phosphocreatine, 200 units/ml phosphocreatine kinase, 10 g/ml of the appropriate M13mp (form I) DNA substrate, and 0.5 mg/ml cell-free extract. Secondary screening of each mutant plaque was conducted in preparation for PCR amplification of the lacZ␣ gene [58] and subsequent nucleotide sequence analysis that was conducted by the Center for Gene Research and Biotechnology (Oregon State University)
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