Azidothymidine and other chain terminators are mutagenic for template-switch-generated genetic mutations

Abstract

The accumulation of mutations causes cell lethality and can lead to carcinogenesis. An important class of mutations, which are associated with mutational hotspots in many organisms, are those that arise by nascent strand misalignment and template-switching at the site of short repetitive sequences in DNA. Mutagens that strongly and specifically affect this class, which is mechanistically distinct from other mutations that arise from polymerase errors or by DNA template damage, are unknown. Using Escherichia coli and assays for specific mutational events, this study defines such a mutagen, 3'-azidothymidine [zidovudine (AZT)], used widely in the treatment and prevention of HIV/AIDS. At sublethal doses, AZT has no significant effect on frame shifts and most base-substitution mutations. AT-to-CG transversions and deletions at microhomologies were enhanced modestly by AZT. AZT strongly stimulated the "template-switch" class of mutations that arise in imperfect inverted repeat sequences by DNA-strand misalignments during replication, presumably through its action as a chain terminator during DNA replication. Chain-terminating 2'-3'-didehydro 3'-deoxythymidine [stavudine (D4T)] and 2'-3'-dideoxyinosine [didanosine (ddI)] likewise stimulated template-switch mutagenesis. These agents define a specific class of mutagen that promotes template-switching and acts by stalling replication rather than by direct nucleotide base damage.

Fig. 1.

Quasipalindrome-associated template-switch mutations. (A) Example of a quasipalindrome with imperfect inverted symmetry; boxed residues are unpaired and targets for mutagenesis. (B) An intramolecular template-switch in a hairpin structure. Leading and lagging strand synthesis target different residues for mutation. A second “restoration” template-switch resumes replication, producing a premutational mispair. (C) An intermolecular template-switch across the replication fork. (D) Mutational reporters in lacZ, QP5 and QP6, detect template-switch generated Lac⁺ revertants on the leading and lagging strand, respectively.

Fig. 2.

Stimulation of templated mutations by AZT. Mutation rates without (white bars) and with 3 ng/mL (gray) or 10 ng/mL (black) AZT. Mutational events include a templated 4-bp frameshift on the leading strand (QP5) and lagging strand (QP6) (A) or a 11-bp microhomology deletion assayed in wild-type (wt) and lexA3 strain backgrounds (B). The higher dose could not be used in lexA3 experiments, because of enhanced lethality and selection for genetic suppressors. Error bars represent 95% confidence intervals for mutation rates, mutations per cell generation.

Fig. 3.

Disk diffusion assay for Lac reversion using QP5 or QP6 and lactose X-gal papillation medium. Each disk on the right was impregnated with 40 μL of the drug (AZT, ddI, or D4T) at the indicated concentration whereas the control disk on the left was spotted with 40 μL of water.

Fig. 4.

Effect of AZT on base substitutions (A) and frameshift mutations (B). Shown are rates for each specific mutation in mutS strains, without (gray bars) and with 10 ng/mL (black) AZT. Error bars represent 95% confidence intervals for mutation rates, mutations per cell generation.