Assay for Quantification of 8Oxoguanine Removal from Plasmid DNA in Human Cells

A high mutation rate in some specific cells should correspond to a defect in removal of 8-oxoG in those cells. To examine this possibility, we constructed vectors having deletions of the SV40 origin of replication (Fig. 1), which therefore cannot replicate in human cells.5

Construction of Closed Circular Nonreplicating Plasmids Carrying Unique 8-Oxoguanine Lesion

Plasmid DNA deleted of the SV40 replication origin (pS ASVori) is constructed as previously described.4 Briefly, plasmid DNA is digested with PvmII, and the Pvull fragment is replaced with an identical fragment deleted of the SV40 origin of replication derived from the pLAS-wt plasmid, a generous gift from L. Daya-Grosjean (UPR 2169, CNRS, Villejuif, France).12 The resulting plasmid, incapable of replicating, is able to transcribe the lesion by using the early promoter when transfected into human cells and is used to construct the monomodified plasmid DNA according to the protocol described above.

Plasmid deleted of its early region promoter (pSApeariy) is constructed by digesting single-stranded plasmid with HaeIII and Pvull enzymes after hybridization of specific oligonucleotides able to hybridize and produce a short double-stranded DNA sequence at the restriction sites. The oligonucleotides are removed by heating at 90° for 2 min, followed by filtration through a Spun size S-400 column (Pharmacia). The longest restriction fragment is gel purified, using a Nucleotrap purification kit (Clontech, Montigny-le-Bretonneux, France) and recircularized, using 800 units of bacteriophage T4 DNA ligase for 4 hr at 16° to produce double-stranded pS 189 ApeariyGO: C deleted of 285 bp. This vector has therefore also lost its SV40 replication origin (Fig. 1) and can neither replicate nor transcribe either T antigen or the lesion-containing oligonucleotide inserted into its 3' untranslated region (UTR).

Quantification of 8-Oxoguanine Repair in Human Cells

Transfection of the nonreplicating GO: C plasmids with or without the early promoter deletion is performed as described for the mutagenesis assays. Cells are collected for recovery of extrachromosomal plasmid DNA after periods of incubation ranging from 2 to 72 hr. Elimination of any contaminating extracellular input DNA is performed by treatment of cell cultures with DNase I before extraction. Recovered plasmid molecules are individualized and amplified by transformation of the Escherichia coli PR195 mutY~/fpg~ strain (Alac-pro F' pro lacl lacZ

12 L. Daya-Grosjean, M. R. James, C. Drougard, and A. Sarasin, Mutat. Res. 183, 185 (1987).

mutY::kanfpg::kan Tn/0), a generous gift from S. Boiteux (CEA, Fontenay-aux-Roses, France), to amplify the S V40 on-deleted plasmid progeny and to avoid any repair of GO: C mispairs in the bacteria. Plasmid DNA from individual colonies is analyzed for repair of the lesion in human cells by digestion with NgoMYV. Persistent 8-oxoG lesions in plasmids not repaired in human cells give rise to mutations in a fraction of the progeny in a given clone after replication in the repair-defective bacteria, thus giving a fraction of plasmid molecules in that clone that is resistant to digestion. In contrast, repair of the lesion in human cells yields clones after passage through the bacteria in which all the progeny DNA is digested by NgoMlV. In good general agreement with estimates of the frequency with which A is inserted opposite 8-oxoG in E. coli,13 approximately 20-30% of the plasmid molecules in the mixed clones is uncut. Repair of the lesion in human cells is calculated as a ratio of the number of colonies in which all progeny molecules are sensitive to NgoMlV digestion to the total number of colonies analyzed. As a control for each experiment, this value is normalized to that found after direct transformation of bacteria with the original GO: C-containing vector. The result for each time point is quantified from three or four independent transformations, with approximately 100 colonies analyzed for each transformation.

Results for removal of 8-oxoG obtained using these plasmids agree very well with those obtained for mutagenesis frequency with replicating plasmids. Thus, the cells that induce high frequencies (30-40%) of G-to-T transversion at GO exhibit almost no repair of the lesion, whereas control cells with a low mutation rate repair almost 100% of GO in 12 hr.5 Interestingly, all human cell lines known to be deficient in the transcription-coupled repair (TCR) of UV-induced DNA lesions, that is, Cockayne syndrome (CS) and combined CS and xeroderma pigmentosum (XP/CS) patients, are deficient in GO repair, but only when the lesion is located in a transcribed sequence.514

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