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 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 α, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mp19, single-stranded DNA constructs were isolated, and the progression of synthesis by polymerase α 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 oncogene. 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 involving codons 12 and 13 may contribute to the pausing of DNA polymerase α and to the generation of clustered mutations at this site.

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This work was supported by grants from the National Cancer Institute (Outstanding Investigator Grant R35-CA-3990 to L. A. L.), the Association pour la Recherche contre le Cancer (J. S. H.), the U.S.-Israel Binational Science Fund, the Basic Research Fondation administered by the Israel Academy of Sciences and Humanities, and the Israel Cancer Association through the late Judith Klienberger Fund (M. F.).

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