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Abstract Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1,000 times greater than during replication. Many mutations involve dissociating a partially copied strand from its repair template and re-aligning with the same or another template, leading to −1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura−) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand. 

DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations. 

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-Azacytidine (5-azaC) is a potent mutagen for template-switching. This effect is dependent on DNA cytosine methylase (Dcm), implicating the Dcm-DNA covalent complex trapped by 5-azaC as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, which can be explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require double strand break repair via RecABCD; the ability to induce the SOS response is anti-mutagenic. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching, and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching with similar genetic properties. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.