Site- and strand-specific mismatch repair of human H-ras genomic DNA in a mammalian cell line

Abstract

Defective mismatch repair has recently been implicated as the major contributor towards the mutator phenotype observed in tumour cell lines derived from patients diagnosed with hereditary non-polyposis colon cancer (HNPCC). Cell lines from other cancer-prone syndromes, such as xeroderma pigmentosum, have been found to be defective in nucleotide excision repair of damaged bases. Some genetic complementation groups are defective specifically in transcription-coupled excision repair, although this type of repair defect has not been associated with cancer proneness. Mechanisms contributing to the high incidence of activating point mutations in oncogenes (such as H-ras codon 12) are not understood. It is possible that novel mechanisms of misrepair or misreplication occur at these sites in addition to the above DNA repair mechanisms. In this study, we have compared the rate of strand-directed mismatch repair of four mispairs (G:A, A:C, T:C and G:T) at the H-ras codon 12, middle G:C position. Our results indicate that, although this location is not a 'hot spot' for bacterial mismatch repair, it is a 'hot spot' for decreased repair of specific mismatched bases within NIH 3T3 cells. NIH 3T3, unlike Escherichia coli, have an extremely low repair rate of the G:A mispair (35%), as well as the A:C mispair (58%) at this location. NIH 3T3 also have a moderately low repair rate of the T:C mispair (80%) at the codon 12 location. Conversely, NIH 3T3 repair of G:T (100%) is comparable to E. coli repair (94%) of this mismatch. These results demonstrate that a mismatch containing an incorrect adenine on either strand at the H-ras codon 12 middle base pair location is most likely to undergo a mutational event in NIH 3T3 cells. Conversely, a mismatch containing an incorrect thymine in the transcribed strand is least likely to undergo a mutational event.

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

“…Previously, we demonstrated significant differences in repair rates for different mismatches at the middle nucleotide position of codon 12 within replicating NIH 3T3 cells (33). All clones in which the mismatch at codon 12 had not been correctly repaired to wild-type (G:C) contained a mixture of wild-type and mutated base pairs (G:C and T:A or A:T depending on the mismatch used).…”

“…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.…”

“…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.…”

“…As shown, Ͼ96% of the cells were in G 1 throughout the 5 h transfection process and for 7 h post-transfection. Analysis of in vivo mismatch repair by individual cells was performed by selecting for the growth of hygromycin-resistant colonies, subsequent PCR amplification of a 128 bp segment from the plasmid containing H-ras codon 12 and analysis by restriction digest and DNA sequencing to determine the sequence at codon 12 (33,34).…”