DNA Polymerase Mechanism
Nucleotides are essential elements of life as they store the genetic code. Because they store the genetic code they must be duplicated with great care. The enzyme responsible for this replication mechanism is called DNA polymerase (Fig 1). There are at least 15 different DNA polymerases operating in our body, with functions that include replicating DNA in cell nuclei or in mitochondria and repairing or bypassing DNA damage.
Fig 1. Conformational change of HIVRT, an enzyme responsible for DNA synthesis in HIV virus, when an incoming nucleotide binds to the growing DNA strand.
The general mechanism of replication involves more steps than the chemical reaction. The complete cycle of the process involves incoming nucleotide binding leading to the selectivity loop closing that forms the pre-catalytic complex. Followed by the chemical reaction and pyrophosphate (PPI) group releases from the active site. The duplicated DNA then translocate one unit that leads the trigger loop opening and completing one nucleotide synthesis.
What makes us to study the polymerase is its high fidelity. We can define fidelity as the rate of correct product formation over all other possible products. The frequency of making an error varies 10-4 for the sloppiest polymerase to 10-8 to a precise one. 10-8 means the enzyme makes one mistake for every 108 reactions. Considering the very many incorrect substrates present in solution, this is quite interesting.
Even more interesting is that those polymerases that are responsible for replicating DNA are the fastest and most accurate, whereas repair enzymes are the slowest and least accurate. Yet it is still not clear why the fidelity varies from one enzyme to the other even though the reactants and products be the same. To study this interesting problem, we use a theoretical approach called Molecular dynamics (MD) simulations. MD is a method that can provide all in one framework therefore potentially be very useful to study the polymerases as well as other biological processes.
In Molecular Dynamics Simulation we solve the classical equations of motion of atom positions in the enzyme and we obtain the coordinates and velocities of each atom as a function of time. Using proper setup, and enough sampling the coordinates as a function of time provides equilibrium positions of atoms. The snapshots in time can also be used to study structure, dynamics as well as the kinetics and thermodynamics of the system at hand.
The biggest challenge in studying biological processes with MD is the time scale gap. MD is limited to time scales in the range of nano-microsecond that puts a limitation into its usage in biology as the processes often times extends to sub seconds.
The method we have chosen to solve the timescale problem is called Milestoning. Milestoning is an algorithm and theory to extend the timescale of MD simulation without giving up the all atom detail. In Milestoning we partition the space into milestones and we use short molecular dynamics trajectory fragments running between milestones to estimate the overall rate in a non-Markovian model.
Milestoning allows extending the timescale of MD simulations to seconds allowing to study the rate of conformational transitions in DNA polymerase (1,2).
Fig. 1 (above) shows the open to closed conformational transition studied by MD simulations with the rate of the process estimated by Milestoning.
References
1-Kirmizialtin, S., Nguyen, V., Johnson. K. A., Elber R., “How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations”, Structure, 20, 618-627, (2012)
2-Kirmizialtin, S., Johnson KA, Elber, R.," Enzyme Selectivity of HIV Reverse Transcriptase: Conformations, Ligands, and Free Energy Partition", J. Phys. Chem. B, 119, 11513, (2015)