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

Abstract

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 U⅐A, U⅐G, and U⅐T targets contained within the lacZ␣ gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase ␤ (؉/؉) and (؊/؊) cellfree extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (ϳ88%) 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 ϳ20% 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 ϳ2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U⅐T) DNA was determined to be 5.7-7.2 ؋ 10 ؊4 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 (U⅐G) 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 U⅐G target and frequently involved a 1-base deletion or incorporation of dTMP.

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1

“…Since DNA ligase I also interacts with polb, p21 can interfere with polb-DNA ligase I interaction; thus, the completion of BER. In a recent study, a similar view concerning the role of p21 in polb-dependent BER has been associated with DNA repair patch size of the natural AP site-mediated BER (Bennett et al, 2001). While this manuscript was in preparation, another report using double-stranded linear DNA substrate demonstrated that p21 inhibits LP-BER in vitro by disrupting PCNA-directed stimulation of FEN1, DNA ligase I, and DNA polymerase d (Tom et al, 2001).…”

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

“…Bacteriophage M13mp2 was propagated in JM109 and M13mp2 single-stranded DNA was purified using the CTAB method as previously described [45]. Oligodeoxynucleotide U-23-mer, which contained a single uracil residue corresponding to position 80 of the E. coli lacZα gene, was 5′-end phosphorylated and annealed to M13mp2 single-stranded DNA to create a site-specific U·A base pair, and a primer extension reaction was conducted as previously described [46].…”

“…1. In order to construct a uracil-DNA standard, double-stranded M13 mp2 DNA that contained a site-specific U·A base pair was prepared by annealing a uracil-containing oligodeoxynucleotide to single-stranded M13 DNA and conducting a primer extension reaction as previously described [45]; this M13 U·A DNA contained one uracil residue per 14,392 nt. First, the M13 U·A standard DNA and a highly purified sample of the unknown DNA to be analyzed were pre-treated in separate reactions with Nfo, Pol β 8 kDa domain dRPase, and methoxyamine (MX) in order to remove ARP-reactive sites.…”

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

“…The primer extension reaction was incubated at 0 °C for 5 min, 22 °C for 5 min, and 37 °C for 3 h, and was terminated by the addition of EDTA to 10 mM. Form I and Form II M13 [uracil-3 H] DNA were isolated by cesium-chloride ethidium bromide density centrifugation as described by Bennett et al [33]. Form III M13 [uracil-3 H] DNA (linear DNA) was prepared from Form II M13 [uracil-3 H] DNA by EcoRI restriction endonuclease digestion.…”