Requirements for frameshift (deletion) during translesion synthesis

Abstract

Frameshift (deletion) is induced by many types of DNA damage in cells. However, the mechanism by which deletions are generated has not been extensively explored. The number of deletions during DNA synthesis catalyzed by the 3’→5’ exonuclease-free (exo-) Klenow fragment of Escherichia coli DNA polymerase I (pol I) was determined systematically on dG-acetylaminofluorene (dG-AAF)-modified oligodeoxynucleotides templates with different bases 3’ and/or 5’ to the lesion. Under conditions where the dNMP (deoxynucleoside 3’-monophosphate) positions opposite dG-AAF can pair with its complementary base at the 5’flanking position, one-base deletions are produced. Since the relative frequency of base insertion opposite the lesion followed the order: dCMP>dAMP>dGMP>dTMP, frequency of generating deletions paralleled to the insertion frequency of dNTP opposite the lesion. Inhibition of chain extension from the dC:dG-AAF pair also be involved in the formation of deletions. These results were supported by steady-state kinetic studies. Two and more base deletions were formed in a similar manner to that observed for one-base deletions. When the dG-AAF-modified templates containing iterated bases 5’ to the lesion were used, shorter deletions predominated. The formation of deletions was reduced when exo+ Klenow fragment was used, suggesting that the proofreading function of the enzyme minimizes the deletion formation. Thus, the ability of generating deletions depends on the (a) the nature of base inserted opposite the lesion, (b) sequence context to the lesion, and (c) the overall rate of translesion DNA synthesis past the lesion. The mechanism for deletions by E. coli DNA pol I may be applied to predict the nature of deletions generated by a variety of DNA adducts and by other prokaryotic and eukaryotic DNA polymerases.