Scientists have long wondered how the initial chemical reactions of life began and why some reactions, like that for DNA polymerization, persisted while others did not. In fact, the specific chemical reaction that DNA and RNA polymerase enzymes catalyze is conserved across all known biological systems, and variations on that reaction were unknown until recently.
The native DNA polymerase reaction involves formation of a bond between the 3′ hydroxyl group on the growing DNA strand and a phosphate group on a nucleoside triphosphate that is paired to the template DNA strand to form a phosphodiester bond (O3′-P5′ linkage). Classically, this requires the help of two metal ions that stabilize the reaction’s transition state. If the 3′ group on the growing end is anything but a hydroxyl, the chain terminates and the reaction doesn’t go any farther.
However, with the wide variation observed in nature, biologists have wondered why there aren’t polymerases that have mutated to catalyze other reactions and, if they exist, could they be valuable for creating synthetic pieces of DNA that could be used in medicine or for other purposes? Recent work conducted at the National Institute of General Medical Sciences and National Cancer Institute Structural Biology Facility (GM/CA) beamline 23-ID-B of the Advanced Photon Source by a team of researchers from Harvard, Massachusetts General Hospital, and the University of Chicago found some answers to these questions that could both move the field of directed evolution of DNA polymerases toward development of new treatments and improve our understanding of the origins of life at the same time. The APS is a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory.
The research was a deeper dive into the team’s previous work that identified a DNA polymerase from a soil bacterium (Geobacillus stearothermophilus) that can be modified with a mutation that allows it to add a new nucleotide to a growing 3′ DNA end with a terminal amine group, forming an unnatural phosphoramidate bond (N3′-P5′ or NP bond) rather than the canonical phosphodiester bond (Figure 1A). The mutated enzyme is much slower than the native polymerase and, in their initial work, seemed to require only one metal ion for catalysis.
In order to understand this mutated enzyme in more detail, the researchers conducted time-resolved X-ray crystallography on the enzyme in action. First, a mixture of the enzyme with the appropriate reactants for NP bond formation was crystallized under conditions in which the reaction does not proceed. Then, crystallized polymerase complexes were activated to catalyze the reaction using a pH shift from 6 to 8.8 and allowed to run for different time points between 0 and 24 hours before being flash frozen to stop the reaction (Figure 1B). They collected data for 19 crystals at six different time points and analyzed data for up to four crystals at each time point (Figure 1C). The unnatural NP bond can be seen accumulating in the resulting structures in the presence of only a single metal ion cofactor.
For the natural DNA polymerase reaction, the transfer of two protons can be detected in the rate limiting step. In order to understand whether the mutated bacterial enzyme has the same constraints, the team conducted solution phase kinetics experiments to look at the proton transfer steps in that catalytic reaction. The kinetics suggested that proton transfer is not the rate limiting step for the NP reaction but that the chemical step involving only a single metal ion, rather than the classical pair of metal ions, is more important for the rate of the reaction. In the natural polymerase, the metal ions stabilize the transition state during the reaction so that the reactants can maintain the optimal orientation and charge distribution needed to proceed.
The researchers hypothesized that, with only one metal ion involved, perhaps this stabilization effect was weakened. They decided to try adding trivalent rare earth and post-transition metal ion cofactors with different properties and this led them to a series of trivalent cations that could speed up the reaction by up to 100-fold, enhancing the production of NP-linked DNAs vastly longer than those produced by chemical synthesis. Armed with unnatural linkages, these DNAs could be used in developing new therapeutics that resist the natural mechanisms of destruction that recognize unmodified DNA and RNA.
The team is hoping to take these findings a step further by harnessing this new enzyme activity to study how the behavior of NP-linked DNA compares to the natural DNA and RNA materials that form the genetic basis for life. – Sandy Field
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See: V.S. Lelyveld1, Z. Fang1,2, and J.W. Szostak1,2,3,4, “Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity”, Science 382, 6669, 423-429 (2023)
Author affiliations: 1Harvard Medical School; 2University of Chicago, 3Massachusetts General Hospital, 4Harvard University
This work was supported by the Howard Hughes Medical Institute and Simons Foundation (grant 290363 to J.W.S.). The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The ALS is a Department of Energy (DOE) Office of Science User Facility supported by contract no. DE-AC02-05CH11231. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health (NIH), National Institute of General Medical Sciences (grant P30 GM124169). GM/CA@APS has been funded by the National Cancer Institute (grant ACB-12002) and the National Institute of General Medical Sciences (grant AGM-12006, P30GM138396). This research used resources of the APS, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The Eiger 16M detector at GM/CA-XSD was funded by NIH grant S10 OD012289.
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