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 basesubstitution 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.] is a frontline drug in the treatment of HIV/AIDS. Its therapeutic effects arise by its incorporation during HIV reverse transcription, resulting in chain termination. Azidothymidine is genotoxic, particularly to mitochondria, presumably because mitochondrial DNA polymerase gamma readily incorporates AZT during DNA synthesis (reviewed in Ref. 1). However, AZT also induces formation of micronuclei, sister chromatid exchange events, and various chromosomal aberrations, suggesting it may also be incorporated into nuclear DNA at some level (2). Using the bacterium Escherichia coli as a model, we have shown that AZT blocks DNA replication and causes formation of single-strand DNA gaps and double-strand breaks (3). E. coli cells appear to tolerate a certain level of AZT by the combined action of the DNA damage response, homologous recombination, and exonuclease excision. The chain-terminating azidothymidine monophosphate does not appear to be removed by intrinsic DNA polymerase proofreading; rather, the exogenous 3′ to 5′ dsDNA exonuclease, exonuclease III (an ortholog of human APE1), is likely the enzyme that removes the residue from DNA during sublethal exposure, because ExoIII − (xthA) mutants are highly sensitive to the drug and have an enhanced DNA damage response (3). A 75 ng/mL dose of AZT reduces viability of xthA mutants about 10,000-fold; because this dose has negligible effects on wild-type cells, many AZT-monophosphate lesions can be removed by ExoIII to sustain replication and proliferative capacity.In this study we investigate the mutagenicity of AZT, at chronic, sublethal doses ∼100-fold ...

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

“…This activity increases the frequency of template-switch-generated mutations in E. coli, which are conceptually similar to the template-switching events proposed by our model (28). Switching provoked by AZT would therefore be expected to increase the frequency of RecA-independent recombination and, if it is frequent enough, could reduce the overall size of genomic exchanges.…”

“…It is known to promote template switching during replication (28). We earlier suggested the involvement of template switching to explain recombination in our system (23).…”

Template-switching during replication fork repair in bacteria

“…Certain DNA damaging agents have been shown to be mutagens for QPM using the lacZ reporter systems; AZT is a particularly strong QPM mutagen, with no significant effect on base substitutions or frameshift mutations [121]. This is consistent with the idea that persistent nascent strand 3′ ends, promoted by AZT incorporation, are more likely to be displaced and to template-switch.…”

Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans

“…Indeed, stalled forks have recently been shown to produce DSB-independent fusions of nearby inverted-repeats, which lead to the formation of palindromic chromosomes (Mizuno et al 2009;Paek et al 2009). The mechanisms by which these fusions take place remain a subject of debate, and three distinct possibilities have been proposed: faulty template switching, tandem inversion duplications, and replication U-turns (Mizuno et al 2009(Mizuno et al , 2013Paek et al 2009;Kugelberg et al 2010;Seier et al 2012).…”