Alkylation-induced frameshift mutagenesis during in vitro DNA synthesis by DNA polymerases alpha and beta.
We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. 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 beta and polymerase alpha-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 alpha-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 beta 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 beta. 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.
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The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, USA. kae4@psu.edu
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D000477:Alkylating Agents / Q000494:pharmacology*; D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000494:pharmacology; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018139:Simplexvirus / Q000201:enzymology; D013379:Substrate Specificity; D013698:Templates, Genetic; D013937:Thymidine Kinase / Q000235:genetics
D000477:Alkylating Agents; D004247:DNA; D013937:Thymidine Kinase; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D005038:Ethylnitrosourea