Abstract 2219: Development of an assay to detect transcription misincorporation errors in Escherichia coli

Abstract

Abstract In contrast to errors in DNA replication which can product heritable mutations, errors in transcription cause only transient alterations. The temporary nature of these transcription mistakes has made them difficult to detect and genetic screens to identify mutations that lower the fidelity of transcription have been problematic. We addressed this problem by developing methods to turn transient transcription errors into permanent genetic changes. The principle is based on suppression of a mutation in the active site of the cre recombinase. This assay is based on two parts: a source of Cre, which is actually the transcription misincorporation substrate, and a genetic reporter with a phenotypic change created by Cre-mediated recombination between lox sites. To develop a substrate for E. coli we chose the galactokinase gene (galK) as our reporter. From an alignment of the galK protein from E.coli, H. sapiens and L. lactis we identified sites that might be able to accept a 12aa insertion encoded by the LoxP sequence without disrupting its function. We selected four different sites that were away from the GalK active site and on the surface of the protein. We started our project using the strain MG1655, which is galK (+), able to grow in galactose as a carbon source and red on a MacConkey galactose plate (MAcGal). Using oligo recombineering, we first inserted a 7 base sequence, which caused a frame-shift mutation, the cells became galK (-) and, therefore white on MacGal. To put it back in frame, we next replaced the 7 bases with a 36 base loxP site, which, if it does not disrupt the function of galK will make the cells galK (+) again. To our surprise, the insertion of this LoxP site was completely successful, putting the gene back in frame and making the cells galK (+) and, therefore, red at each of the four sites we chose. A derivative of this reporter was made with the a portion of the galK gene inverted at one of the loxP sites. Active Cre recombinase can be detected by flipping the inversion to restore galK function. The active site mutant cre-Y324C exhibits a very low inversion rate. Mutations that reduce the accuracy of transcription, such as cells defective in greA, exhibit an elevated frequency of galK (+) inversions. This system will be used to identify additional mutations that reduce the fidelity of transcription in E. coli. Citation Format: Jorge A. Irizarry-Caro, Mary Ernst, Carolyn Court, Alison Rattray, Mikhail Bubunenko, Ding Jin, Donald Court, Jeffrey Strathern. Development of an assay to detect transcription misincorporation errors in Escherichia coli. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2219. doi:10.1158/1538-7445.AM2015-2219