Progression of DNA Synthesis Catalyzed by DNA Polymerase Protooncogene Interrupt the ras Codons 12 and 13 of H - Updated

Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in the H-ras gene are predominantly single-base substitutions and are most fre quently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for raj mutation with respect to abnormalities in copying by purified DNA poh merase a, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mpl9, single-stranded DNA constructs were iso lated, and the progression of synthesis by polymerase a was measured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the middle base of codon 12, the same alteration that converts H-ras into an activated onco gene. Resistance of codon 12 in double-stranded constructs 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 DNA chains that have been extended past codons 12 and 13 was found to be <0.01. We consider a variety of mechanisms by which the potential secondary structure in volving codons 12 and 13 may contribute to the pausing of DNA poly merase a and to the generation of clustered mutations at this site.