CODON-12 AND CODON-13 OF H-RAS PROTOONCOGENE INTERRUPT THE PROGRESSION OF DNA-SYNTHESIS CATALYZED BY DNA POLYMERASE-ALPHA

Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in t he H-ras gene are predominantly single-base substitutions and are most frequently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for ras mutation with respect to abnormalities in copying by purified DNA polymerase a, a major eucaryotic replication enzyme. Exon 1 of H-r as gene was inserted into M13 mp19, single-stranded DNA constructs wer e isolated, and the progression of synthesis by, polymerase a was meas ured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the midd le base of codon 12, the same alteration that converts H-ras into an a ctivated oncogene. Resistance of codon 12 in double-stranded construct s to digestion with restriction enzymes and computer investigation of the ras sequence suggest that these termination sites are in a region of secondary structure. The frequency of sequence alterations within D NA chains that have been extended past codons 12 and 13 was found to b e <0.01. We consider a variety of mechanisms by which the potential se condary structure involving codons 12 and 13 may contribute to the pau sing of DNA polymerase a and to the generation of clustered mutations at this site.