Mutational specificity of depurination

The mutagenic consequences of damage to DNA produced by low pH and high temperature have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When damaged single-stranded DNA from bacteriophage M13mp2 is used to transfect competent Escherichia coli cells, a 15-fold increase in mutation frequency, measured as loss of alpha-complementation by the lac DNA in the phage, is observed compared with an untreated DNA control transfection. The enhanced mutagenicity is largely dependent on induction of the error-prone SOS response and is proportional to the number of lethal hits introduced into the DNA. The effect is abolished by treatment of the damaged DNA before transfection with either apurinic/apyrimidinic endonuclease or alkali. Based on these observations and the rate constants for formation of the known heat/acid-produced lesions in DNA, it is concluded that the majority of the induced mutagenesis results from apurinic sites. DNA sequence analysis of 87 spontaneous and 124 induced mutants indicates that the major effect is on single base-substitution mutagenesis with a small increase in (deletion) frame-shift frequency. Approximately 80% of the base-substitution mutations occur at purine positions in the viral strand, consistent with depurination as the predominant premutagenic lesion. The preference of guanine over adenine sites mutated is consistent with the preference for depurination of guanine over adenine. Transversions are observed for 57 of 79 (72%) induced base substitutions, with a strong preference for insertion of adenine residues opposite the putative apurinic site. These data in a forward mutational system provide insight into the mechanisms used by a cell to replicate DNA containing noncoding lesions.