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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.