On the mechanism of frameshift (deletion) mutagenesis in vitro

Abstract

An experimental system has been developed to quantify frameshift deletions and base substitutions formed during DNA synthesis in vitro. Oligodeoxynucleotides, modified site-specifically with acetylaminofluorene or other adducts and lesions, were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The influence of DNA sequence context on frameshift mutagenesis was determined by modifying systematically the bases flanking the lesion. Frequencies of nucleotide insertion opposite the lesion and chain extension from the 3'-primer terminus were established by steady state kinetic analysis. The ability of a damaged nucleotide to generate one-base and two-base frameshift deletions was determined primarily by two parameters: the nature of the base inserted opposite the adduct with respect to the sequence context in which the lesion is embedded and the overall rate of translesional DNA synthesis. Frameshift deletions generated during DNA synthesis were greatly enhanced in the absence of proofreading exonuclease. Misinsertion of bases opposite the lesion precedes misalignment of the template-primer. Extending on earlier studies (Kunkel, T. A. (1990) Biochemistry 29, 8003-8011), a model has been proposed and used in various sequence contexts to predict the propensity of aminofluorene adducts, exocyclic DNA adducts, 8-oxopurines, and synthetic abasic sites to generate frameshift deletions in vitro.