Tautomerisation Mechanisms in the Adenine-Thymine Nucleobase Pair during DNA Strand Separation

The adenine-thymine tautomer (A*-T*) has previously been discounted as a spontaneous mutagenesis mechanism due to the energetic instability of the tautomeric configuration. We study the stability of A*-T* while the nucleobases undergo DNA strand separation. Our calculations indicate an increase in the stability of A*-T* as the DNA strands unzip and the hydrogen bonds between the bases stretch. Molecular Dynamics simulations reveal the time scales and dynamics of DNA strand separation and the statistical ensemble of opening angles present in a biological environment. Our results demonstrate that the unwinding of DNA, an inherently out-of-equilibrium process facilitated by helicase, will change the energy landscape of the adenine-thymine tautomerization reaction. We propose that DNA strand separation allows the stable tautomerization of adenine-thymine, providing a feasible pathway for genetic point mutations via proton transfer between the A-T bases.

The process of the tautomerisation reaction of adenine-thymine (A-T) is detailed in the manuscript. A broad overview of the tautomerisation process is presented in Supplementary Figure 1.
Once the non-standard forms of the bases, A* and T*, are established on isolated DNA strands during mitosis, the next step in the biological process is the formation of the new DNA strands by adding nucleotides at the active site of the DNA polymerase. The mutated bases A* and T* can bond in the non-standard, Watson-Crick-like pairings A*-C and G-T*. These pairings are shown in Supplementary Figure 2.
The consequence of the non-standard base pairings is that an A*-C pair is created in one strand of DNA and a G-T* pair in another instead of two A-T base pairs. These DNA code errors can evade replisome fidelity checkpoints and create equivalent errors through subsequent generations of DNA replication. The tautomerisation of A-T is an asynchronous, step-wise reaction with a zwitterionic intermediate product from a single proton transfer which  [1,2]. These base pairs are incompatible with the double helix structure, so mitosis will cease when the replication machinery encounters zwitterionic products. Therefore, only double proton transfer tautomerisation products are relevant to spontaneous mutagenesis.

SUPPLEMENTARY NOTE 2: MOLECULAR DYNAMICS SIMULATIONS
We use molecular dynamics (MD) to investigate the detail of the DNA strand separation process. We calculate the occurrences of the opening angles θ across the range of DNA strand separation 0.0Å -2.0Å and estimate the speed at which the strand separation occurs. The MD system is constructed of 14 base pairs within a double strand of DNA, initially in equilibrium and an aqueous environment. In the quantum mechanical investigations, the opening angle smoothly increases during strand separation (see Supplementary Figure 3). We examine whether this observation remains true in the biological ensemble in the MD calculation. We study the opening angle in two scenarios: where the B1 bond is the first to open and where the B2 bond is the first to open. We collect a histogram of opening angles and their occurrences across a 75 • range for each scenario of B1 and B2 opening first. We treat a positive angle as opening from the B2 end of the base pair and a negative angle as opening from the B1 end of the base pair.
The software we use to conduct molecular dynamics (MD) simulations is GROMACS 2018 [3]. The system consists of 14 base pairs within a DNA duplex with the base code: T 3 TTGTACGTACAAA 5 . A 2 nm x 2 nm x 2 nm) simulation box is constructed to surround the DNA system with explicit SPCE solvent and, neutralising sodium ions, a CHARMM36 [4] force field is employed for all the MD simulations. We minimise a group of replica systems, equilibrate them over a scale of 50 ps of NVT ensemble over incremental time steps of 1 fs and simulate 10 different separation forces with a maximum force 12 kJ mol −1 nm −1 . The system temperature is maintained, by a Nose-Hoover thermostat, at 310 K, using 0.2 ps as a coupling constant. The data is collected over 66 system replicas. We collect the time series statistics of the two hydrogen bond lengths between the adenine and thymine base pair and pass these statistics through a Savitsky-Golay filter.
We define the opening angle between the bases with the scheme presented in Supplementary