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DNA Synthesis: Flip It and Reverse It

Every time our cells divide, the DNA inside must be copied, or synthesized, accurately to avoid mistakes that could be harmful to our health. Despite the importance of the process, the precise sequence of steps was only hypothesized, with snapshots pieced together in scientists’ best estimate of their order. Now, timestamps have been added to those snapshots, revealing a different order of steps in the DNA synthesis pathway that, if replicated in additional studies, would upend the model in current textbooks.

A team of UC Irvine researchers led by John Chaput prepared more than 700 protein samples, each plunged into liquid nitrogen and frozen at a different stage in the DNA synthesis process. The “time-stamped” samples were then analyzed using protein crystallography at Advanced Light Source (ALS) Beamlines 5.0.2, 5.0.3, and 8.2.1. The combination of structural and temporal data allowed the researchers to identify distinct conformations at specific times and arrange the snapshots in the order of their appearance in the reaction pathway, providing the first unambiguous picture of the process.

The findings counter previous beliefs about DNA synthesis. During the process, a protein called DNA polymerase copies the DNA sequence by adding one genetic letter (A, C, T, or G) at a time to build a new, paired chain. The current model suggests that the polymerase first binds the DNA and then binds to an incoming genetic letter, after which a chemical bond forms between the new chain and the incoming genetic letter before the polymerase moves to the next position on the chain to repeat the steps. However, the new data show that the last two steps are, in reality, reversed: DNA polymerase moves onto the next position before the incoming genetic letter forms a bond with the new chain.

“This was the single biggest surprise,” said Nicholas Chim, a researcher in Chaput’s group and the study’s first author. “Determining how these enzymes work at the molecular level offers a glimpse into biology that could lead to new advances in human health and biotechnology, such as new approaches to DNA sequencing or therapeutics that inhibit DNA synthesis in diseased cells,” he added.

In DNA synthesis, DNA polymerase (dark gray, with residues Y719 and Y714 highlighted in pink) copies the DNA sequence by adding one genetic letter (A, C, T, or G) at a time to build a new, paired chain. In order to capture the distinct conformational changes, the authors artificially slowed down the reaction, which would normally occur within milliseconds. At the start, residue Y719 associates with the template strand highlighted in red, while residue Y714 associates with the primer strand highlighted in orange. The polymerase uses a push-pull process to allow the new genetic letter (highlighted in blue) to be installed onto the end of the primer strand while concurrently moving to the next position (translocation, 1-8 mins). By 120 min, a chemical bond forms between the new genetic letter (blue) and the primer (orange) and the process repeats.

N. Chim, R. Meza, A.M. Trinh, K. Yang, and J. Chaput, “Following Replicative DNA Synthesis by Time-Resolved X-Ray Crystallography,” Nat. Commun. 12, 2641 (2021), doi: 10.1038/s41467-021-22937-z.