DEVELOPMENT AND USE OF AN IN-VITRO HSV-TK FORWARD MUTATION ASSAY TO STUDY EUKARYOTIC DNA-POLYMERASE PROCESSING OF DNA ALKYL LESIONS

We have developed an in vitro DNA polymerase forward mutation assay us ing damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene, The quantitative method uses compleme ntary strand hybridization to gapped duplex DNA molecules and chloramp henicol selection, This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We ha ve examined the accuracy of DNA synthesis catalyzed by calf thymus pol ymerase alpha-primase, polymerase beta and exonuclease-deficient Kleno w polymerase, Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repe at sequence within the HSV-tk locus, Treatment of the DNA template wit h N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar d ose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-fk system is unique in tha t damage-induced mutagenesis can be analyzed both quantitatively and q ualitatively in human cells, in bacterial cells and in in vitro DNA sy nthesis reactions at a single target sequence.