SITE-SPECIFIC AND STRAND-SPECIFIC MISMATCH REPAIR OF HUMAN H-RAS GENOMIC DNA IN A MAMMALIAN-CELL LINE

Defective mismatch repair has recently been implicated as the major co ntributor towards the mutator phenotype tumour cell lines derived from patients hereditary non-polyposis colon cancer (HNPCC). Cell lines fr om other cancer-prone syndromes, such as xeroderma pigmentosum, have b een found to be defective in nucleotide excision repair of damaged bas es, Some genetic complementation groups are defective specifically in transcription-coupled excision repair, although this type of repair de fect has not been associated with cancer proneness. Mechanisms contrib uting to the high incidence of activating point mutations in oncogenes (such as H-ras codon 12) are not understood, It is possible that nove l mechanisms of misrepair or misreplication occur at these sites in ad dition to the above DNA repair mechanisms, In this study, we have comp ared 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 re sults indicate that, although this location is not a 'hot spot' for ba cterial mismatch repair, it is a 'hot spot' for decreased repair of sp ecific mismatched bases within NIH 3T3 cells, NIH 3T3, unlike Escheric hia 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 l ocation, Conversely, NIH 3T3 repair of G:T (100%) is comparable to E. coli repair (94%) of this mismatch, These results demonstrate that a m ismatch containing an incorrect adenine on either strand at the H-ras codon 12 middle base pair location is most likely to undergo a mutatio nal event in NIH 3T3 cells, Conversely, a mismatch containing an incor rect thymine in the transcribed strand is least likely to undergo a mu tational event.