Antonio Pantelias Garcés

The task of DNA replication is central and essential for all known living organisms. There are many environmental factors that can induce mutations during replication, yet a more subtle mechanism that relies only on the intrinsic properties of DNA was proposed by Watson and Crick. In their seminal 1953 paper describing the double helix, they hypothesised that spontaneous mutations could arise when DNA bases adopt rare and energetically less favourable tautomeric forms. Since tautomerisation involves the relocation of protons, this transition could be facilitated by quantum tunnelling. In this thesis, we describe two experimental strategies to test this hypothesis.

First, we used an indirect way by performing polymerase chain reactions (PCR) of a gene encoding a selectable marker (lacZα) in either normal water (H₂O) or deuterated water (D₂O). The PCR products were cloned in vector plasmids, which were then used to transform E. coli. After plating the transformed E. coli on selective media including X-gal, bacteria containing the wild-type gene formed blue colonies, whereas those containing a mutated copy of lacZα formed white colonies. The mutation rate in each solvent was estimated by counting the ratio of white over blue colonies, known as blue-white screening, resulting in a 2-fold increase in the mutation rate in D₂O ( p < 0.01). However, this approach only provides an approximate estimate of the mutation rate as the coarse-grained sum of all possible types of point mutations induced by a polymerase. In fact, when we sequenced the plasmids from over 100 white colonies, the error rate based on the total analyzed sequence was not significantly different between the two solvents.

Our second experimental strategy employed the direct competition gel fidelity assay, which offers a more precise way to investigate the misinsertion frequency (f_ins) for specific mismatches. In D₂O, the f_ins for dGTP/T and dTTP/G were 2.7-fold and 6.1-fold lower, respectively, than in H₂O. Steady state kinetics experiments confirmed that the f_ins for the same mismatches decrease in D₂O. Interestingly, after calculating the relative polymerase activity for these misinsertions and the corresponding correct insertions, dATP/T and dCTP/G, the impact of D₂O on the f_ins was largely mediated by affecting the activity for misinsertions. The impact of D₂O was 5.3-fold stronger for dTTP/G than for dCTP/G, and 14.3-fold stronger for dGTP/T than dATP/T. This targeted impact indicates that the mechanism involved is not simply a general solvent isotope effect on the polymerase but could be a modification of the tautomerisation equilibria of the DNA bases. While additional experiments need to be performed, these results are consistent with a role for proton tunnelling in the transition from a wobble G·T to a Watson–Crick-like G·T, which preserves the geometry of canonical base pairs and thus facilitates its misinsertion.

Further to the above investigations, as an alternative approach to detecting non-trivial quantum effects in living systems, in this thesis we present experiments to test the hypothesis proposed by the physicist Herbert Fröhlich in the 1960s. He devised a model of energy transfer in biological systems that predicted long-lived and long-range coherent energy states, which could explain the complex organisation observed in biomolecules, like DNA, RNA, and proteins. According to his hypothesis, low-frequency terahertz (THz) modes could be involved in the generation of coherent excitations that may even influence biological processes of higher complexity, such as DNA replication and transcription. In order to explore the interaction between THz radiation and living cells, a global mutagenesis screen as well as a transcriptome-wide analysis of differential gene expression were performed. Our preliminary results indicate that THz irradiation of E. coli cells can lead to non-thermal transcriptional responses.