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 39 exonucleases targeted to the lagging strand. The loss of 39 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 F9 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.

Saccharomyces cerevisiae cells expressing both a-and a-mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MATa. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-D327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Mata2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55D strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MATa cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-D327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-D327 to CPT was unexpectedly increased by the MATa/MATa-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-D327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MATregulated. rad55D is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive. D NA repair in budding yeast is strongly influenced by the cell's mating status. Saccharomyces cells can be of three mating types: those able to mate expressing only MATa or only MATa and nonmating cells expressing both MATa and MATa. MATa/MATa diploid cells are more radiation resistant and recombination proficient than diploids expressing only MATa or MATa (Friis and Roman 1968;Heude and Fabre 1993;Fasullo and Dave 1994;Lowell et al. 2003). A similar increase in radioresistance and resistance to radiomimetic drugs is seen in haploid cells that express both mating-type alleles. Coexpression of MATa and MATa, either in diploids or in haploids, leads to the formation of the Mata1-Mata2 corepressor that turns off the expression of haploid-specific genes and induces expression of diploid-specific genes. The most striking effect of a1-a2 repression is a severe disabling of nonhomologous end-joining (NHEJ) by the repression of NEJ1 (Å strö m et al. 1999;Lee et al. 1999; FrankVaillant and Marcand 2001;Kegel et al. 2001;Ooi and Boeke 2001;Valencia et al. 2001). Whether any of the other targets of a1-a2 repression affect homologous recombination (HR) is not known.A double-strand break (DSB) c...

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 ...