Assay for Mutagenesis of 8Oxoguanine Containing Plasmids Replicated in Human Cells

A sequence containing a unique 8-oxoG was located in a replicating plasmid and inserted either into the 3'-untranslated region of the simian virus 40 (SV40) large T antigen (TAg) gene in the template strand transcribed from the early promoter of SV40 or into a nontranscribed corresponding position on the opposite (sense) strand of TAg. Use of these alternative constructs thus allowed comparison of cellular processing of 8-oxoG with and without transcription of the sequence in which it is located. The shuttle vector replicates autonomously in human cells transfected with the TAg of SV40 because it contains an SV40 origin of replication as well as itself encoding TAg, with essentially all transfected plasmids having replicated by 12 hr after transfection in one cell line studied.4 Replication of the shuttle vector allows determination of the frequency of mutagenesis at 8-oxoG in human cells, which should reflect the efficiency of removal of the lesion (Fig. 1).

Construction of Closed Circular Replicating Monomodified Plasmids

Synthetic oligonucleotides used to construct the monomodified plasmids are from Genset (Paris, France) and are purified in a denaturing 20%(w/v) poly-acrylamide gel. The modified 19-mer oligonucleotide carrying a unique 8-oxoG that we use contains a small fragment of the human Ha-ras gene from codons 10 to 14, with the lesion located on the second guanine of codon 12 (5'-GATC GGC GCC GGOC GGT GTG-3')- The 8-oxoG oligonucleotide is produced as described previously6 by J. Cadet (CEA, Grenoble, France).

3 P. K. Cooper, T. Nouspikel, S. G. Clarkson, and S. A. Leadon, Science 275, 990 (1997).

4 F. Le Page, A. Guy, J. Cadet, A. Sarasin, and A. Gentil, Nucleic Acids Res. 26, 1276 (1998).

5 F. Le Page, E. E. Kwoh, A. Avrutskaya, A. Gentil, S. A. Leadon, A. Sarasin, and P. K. Cooper, Cell 101,159 (2000).

6 V. Bodepudi, S. Shibutani, andF. Johnson, Chem. Res. Toxicol. 5, 608 (1992).

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