Loss of Allosteric Regulation in Ribonucleotide Reductase and its Effect on the E. coli Genome

Abstract

Whilst essential for cell viability, intracellular dNTP pools must be maintained at specific ratios, as well being able to react to rapid changes in demand for dNTPs. This constant regulation of dNTP pool sizes is achieved via allosteric regulation of the enzyme ribonucleotide reductase (RNR), which catalyses the rate limiting step in the synthesis of dNTPs. It has been hypothesised that in early DNA-based organisms, genetically encoded catalytic proteins would have preceded the emergence of ribonucleotide reduction, therefore dNTP pool sizes would not have been affected by RNR allosteric regulation maintaining them in at strict concentrations. Previous studies observed the loss of allosteric regulation in RNR resulting in imbalances of intracellular dNTP pools, leading to increased mutation rates (Ahluwalia & Schaaper, 2013; Kumar, Viberg, Nilsson, & Chabes, 2010). To examine the effects of a loss of allosteric regulation and the loss of RNR, two evolution experiments were attempted. The first involved the insertion of known RNR mutator mutants into an RNR knockout E. coli strain and evolving said strain for 40 daily transfers. The second involved the evolution of an E. coli RNR knockout strain in minimal media supplemented with skewed deoxyribonucleoside (dNS) pools with high concentrations of deoxyadenosine to create an intracellular dNTP imbalance. While we were not able to successfully create the transformed lines for the mutator RNR mutant in the context of this thesis, the evolution experiment with the skewed dNS pool was completed. Results from this experiment showed few mutations overall. This has been attributed to deoxyribonucleotides being used as a carbon source thereby relieving mutational pressure. Whole genome sequencing has shown the same genes acquiring mutations in multiple lines, hypothesised as being fixed as adaptational response to media conditions. Lines grown in high deoxyadenosine concentrations experienced the most mutations with lines acquiring mutations in DNA mismatch repair and synthesis, and with a high skew of SNPs resulting in a change to G/C. These results ultimately indicate that metabolism of excess dNTPs and dNSs as carbon sources in E. coli cells may result in less mutational pressure on the genome.