Alkylation-induced frameshift mutagenesis during in vitro DNA synthesis by DNA polymerases α and β

We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase α-primase and DNA polymerase β. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G→A transition mutations was observed for both polymerases, as expected due to mispairing by O⁶-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase β and polymerase α-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase α-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G→A transition frequency. In the polymerase β reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G→A transition errors by polymerase β. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase. Copyright (C) 1998 Elsevier Science B.V.