Mammalian assay for site-specific DNA damage processing using the human H-rasproto-oncogene

Abstract

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.

Inefficient in vivo repair of mismatches at an oncogenic hotspot correlated with lack of binding by mismatch repair proteins and with phase of the cell cycle

“…Mismatch plasmid construction was performed essentially as described previously (33,34). Briefly, gapped heteroduplex DNA was constructed by restriction 1418 digest of a plasmid containing 2 kb of the H-ras sequence, followed by annealing to single-stranded M13 DNA containing either the coding or noncoding strand of H-ras.…”

“…This procedure produces a plasmid that contains the majority of introduced nicks (four of six) on the same strand as the incorrect base. Under the conditions used, ligation is Ͻ100% efficient and occurs with equal probability on both strands, thus biasing repair to that strand containing the incorrect base (34).…”

“…Nonsynchronous NIH 3T3 mismatch repair experiments were performed as described previously (33,34). For G 1 synchronization experiments, NIH 3T3 cells were seeded at 4.9ϫ10 5 cells per 100 mm plate in media containing 0.25% calf serum and incubated for 3 days at 37°C in 5% CO 2 .…”

“…DNA was purified from each hygromycin-resistant colony and amplified by PCR as described previously (33,34), yielding an amplified plasmid DNA product of 128 bp containing exon 1 of human H-ras plus several human intronic nucleotides, thus precluding amplification of NIH 3T3 genomic DNA. An aliquot of each amplified product was digested with NaeI restriction enzyme for codon 12 analysis or NarI for codon 10 analysis.…”

“…For antibody-binding assays, 1 µg of monoclonal anti-hMSH6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was allowed to incubate with nuclear extracts for 20 min prior to addition of DNA. For ATP inhibition (34) and are shown here for comparison (correct repair: G:A→35%, A:C→58%, T:C→80%, G:T→100%). Repair rates include all mismatches either repaired correctly to G:C or incorrectly to T:A or A:T (in the case of G:T in G synchronized cells, the 100% repair rate reflects 92% correct repair and 8% incorrect repair), but not mismatches which are replicated before repair and thus are found as mixtures in the resulting colony.…”