Photo of Ci Ji Lim
New Images Uncover How Enzyme Kicks Off DNA Replication

University of Wisconsin–Madison researchers and their collaborators have used high-resolution imaging to gain a more nuanced understanding of the structure and multiple functions of an enzyme integral to DNA replication.

As the cells in our bodies replicate, our genetic code is passed from one cellular generation to the next, all while remaining largely unchanged. New strands of DNA are replicated from double-stranded DNA that has started to separate into two separate strands, each called a template strand. The process requires a precise and intricate dance among multiple enzymes and between enzyme sub-units.

Biochemistry assistant professor Ci Ji Lim is interested in learning more about the choreography within one such enzyme: DNA polymerase alpha-primase (Polα-primase for short).

Polα-primase helps to create new strands of DNA by building short strands of genetic material called primers. First, the enzyme attaches to a strand of DNA template and lays down a short length of genetic material called RNA primer. The enzyme then lays down a second length of genetic material, contiguous to the first, called DNA primer. Only when the hybrid RNA-DNA primer is built can DNA replication proceed.

Unlike many other polymerases, Polα-primase lacks the ability to proofread its own work. As a result, although the RNA-DNA primer may contain mistakes, it establishes a point from which more accurate polymerases can finish the job of DNA replication.

Last year, the Lim Lab explored Polα-primase's role in DNA replication at the ends of chromosomes. This year, they investigated the steps by which Polα-primase builds RNA-DNA primers. The new research is a collaboration with the lab of Tahir Tahirov, a professor in the University of Nebraska Medica Center Eppley Institute for Research in Cancer and Allied Diseases.

Scientists now have greater insight into the moment Polα-primase switches from making RNA primer to extending the RNA primer with DNA. The researchers’ latest study, published in Nature Structural Molecular Biology, confirms mechanisms that allow Polα-primase to toggle between its sub-units to build both components of RNA-DNA primer and also provides insights into how the enzyme stops building primer and detaches from the DNA template.

New images of Polα-primase, collected using an advanced biomolecular imaging technique called cryo-electron microscopy (cryo-EM) single-particle analysis, confirm a prevailing hypothesis: the region of Polα-primase responsible for building the RNA primer remains attached to the primer even as the DNA portion of the primer is established.

Lim says these results suggest that the enzyme holds on to the RNA primer while building the DNA primer as a fail-safe.

“Think of a safety harness for a rock climber,” say Lim.

If the DNA polymerase subunit of Polα-primase becomes dislodged before sufficient DNA is added to the primer, the incomplete primer, which remains bound to the enzyme, can rapidly be reengaged by the polymerase.

“We found that Polα-primase continues to hold on to the RNA primer while the enzyme is extending the primer with DNA,” explains Lim. “There have been predictions about this, but we didn’t have direct evidence. Now we’ve caught it in action, and in this case it’s not cliché to say that seeing is believing.”

That’s thanks to cryo-EM, which captures moments in time by rapidly freezing biomolecular samples and stopping them in action. Frozen samples of Polα-primase are then imaged using a transmission electron microscope, and the resulting 2D images — which depict the biomolecules in many different orientations — are reconstructed into high-resolution 3D structures.

The UW–Madison and University of Nebraska Medical Center researchers also hint at how Polα-primase stops building RNA-DNA primer before handing its work off to other components of the DNA replication process.

“There are parts of Polα-primase that control how long the RNA-DNA primer can become,” says Lim. “We found new interactions that are important for making sure that the enzyme lets go after the RNA-DNA primer gets to a certain length.”

These new findings prompt Lim and his collaborators to ask even more questions about how the Polα-primase machinery works during DNA replication. For Lim, a 2022 recipient of an NIH High Risk, High Reward New Innovator Award, this is exciting territory.

“This research is about understanding how genetic information is copied during DNA replication in cells,” says Lim. “We want to know how this multifaceted enzyme acts and interacts through every stage of action.”

Written by Renata Solan.