MAMMALIAN ASSAY FOR SITE-SPECIFIC DNA-DAMAGE PROCESSING USING THE HUMAN H-RAS PROTOONCOGENE

The human genomic H-ras proto-oncogene was inserted into an Epstein-Barr virus (EBV) vector (p220.2) that replicates synchronously with the cell cycle. Unique restriction enzyme sites, 30 bp apart, were created on either side of codon 12 to enable the construction of gapped heteroduplex (GHD) DNA. Depending upon experimental protocol, the gap could be located either on the coding (non-transcribed) strand or the non-coding (transcribed) strand. GHD DNA was created using a 1.8 kb segment of H-ras DNA containing exon 1, into which a synthetic 30 nucleotide oligomer containing a strand- and site-specific mismatched nucleotide was annealed. The 1.8 kb segment of H-ras DNA containing a codon 12; middle G:T, A:C or T:C mismatch has been religated with high efficiency into the EBV vector and transfected into NIH 3T3 cells using a mild liposome-mediated protocol. Subsequent hygromycin resistant NIH 3T3 colonies have been PCR amplified and sequenced. In this study, codon 12; middle nucleotide mismatch correction rates to wild-type G:C during replication in NIH 3T3 cells were 96.4% of G:T mismatches, 87.5% of A:C mismatches and 67% of T:C mismatches.