RESUMO
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.
Assuntos
DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Mutação/efeitos dos fármacos , Zidovudina/farmacologia , Fármacos Anti-HIV/farmacologia , Sequência de Bases , Didanosina/farmacologia , Escherichia coli/genética , Modelos Genéticos , Mutagênicos/farmacologia , Estavudina/farmacologia , Moldes GenéticosRESUMO
Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.