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

The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), double-strand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label abasic sites resulting from uracil excision with biotin.

Evidence for three thymine DNA glycosylases in human cell extracts: substrate specificities of thymine DNA glycosylase activities.

Background: Purified human thymine DNA glycosylase (TDG) recognizes a G: T mispair in a CpG sequence context more strongly than in any other, in addition to its inactivity toward 2-aminopurine: T or 2,6 diaminopurine: T pairs. We investigated the multiplicity of TDG to establish a better relationship between in vitro G: T mismatch incision and in vivo repair of a G: T to a G: C pair.

Material/methods: Cell-free extract was prepared from A1235-MR4 human glioma cells grown in tissue culture.

Multiplicity of strand incision at G:T base mismatches in DNA by human cell extracts.

Cell extract from the HT29 human colon carcinoma cell line (lacking mutator phenotype) was used to study the ATP-dependent G:T mismatch repair. We found that when a 45-bp (model) DNA with a single CpG/TpG mispair was incubated with the cell extract and ATP, it was incised immediately 5' and 3' to the mismatched T, and we noted that the actual 5'- and 3'-labeled fragments were similar to the cleaved products of thymine DNA glycosylase (TDG). This TDG-like cleavage product was enhanced (5-fold) with stimulation of several novel fragments, as inferred from the effect on incision at CpG/TpG site of the addition of G:U competitor DNA and ATP to the HT29 extract.

Substrate specificity and sequence preference of G:T mismatch repair: incision at G:T, O6-methylguanine:T, and G:U mispairs in DNA by human cell extracts.

Extracts of two human glioma cell lines (lacking O6-methylguanine DNA-methyltransferase) (i.e., A1235 and its alkylation-resistant derivative A1235-MR4) were examined for their ability to execute strand incision at different base mismatches in model (45-bp) DNA.