Cryo-EM reveals hidden mechanics of DNA replication and sheds new light on cancer target microbiologystudy

Every day, billions of cells in your body divide, helping to replace old and injured cells with new ones. And each time this happens, your entire genetic library—your genome, which totals more than 3 billion base pairs of DNA—has to be copied, precisely, from the parent cell to the new daughter cell.

When organisms encounter problems—what scientists call “replication stress”—this process is more prone to errors, which often cause mutations in the genetic code. These mutations can be copied forward and give rise to cancer and other diseases.

One source of this stress is when the bio-machinery that does that copying gets physically stuck. And one of the things it can get stuck on is the DNA template itself, which can adopt alternative structures in certain contexts. For example, regions of the genome that are rich in guanine bases (represented by G in the DNA code) can fold into a DNA structure called a G-quadruplex, or G4 for short, which is more compact than normal DNA.

Using cryo-electron microscopy (cryo-EM), a team of structural and molecular biologists at Memorial Sloan Kettering Cancer Center (MSK) set out to investigate G4s—which have gained attention as potential therapeutic targets in cancer—working to understand their influence on DNA replication. Additionally, the scientists unexpectedly captured for the first time a detailed picture of how the “engine” driving the cellular replication machinery moves along DNA in human cells.

Their findings, which were published March 7 in Science, not only reveal new details about how secondary DNA structures like G4s can impede DNA replication, but also offer new insights into fundamental human biology.

The study was led by co-first authors Sahil Batra, Ph.D., a research scholar in the lab of senior author Dirk Remus, Ph.D., and Benjamin Allwein, a graduate student in the lab of senior author Richard Hite, Ph.D.. Both labs are part of the Sloan Kettering Institute, a hub for foundational biology research at MSK.

G-quadruplexes and cancer

“The DNA double helix is one of the most recognizable molecular structures in science,” Dr. Batra says. “But DNA can actually exist in multiple shapes, and G-quadruplexes are one of them. There are drugs being developed to target G4s in cancer cells, but the mechanisms underlying G4s’ harmful effects are not clear—which is one of the reasons we are studying them.”

G4s have been associated with a number of well-known cancer-driving oncogenes like MYC and KRAS, the researchers say, as well as with cancer cells’ ability to extend their lifespans by replenishing their telomeres, the protective caps on their chromosomes.

“So the idea is that by targeting G4s in cancer cells, you can lock them in place, preventing the DNA from being unwound and copied, and thus interfering with the ability of cancer cells to divide and proliferate,” Dr. Remus says. “We’ve known that G4s are associated with genomic instability—and now our study provides a much clearer understanding of how they work and why they’re so detrimental.”

Visualizing G4s in action

Structural biologists use a variety of tools to be able to see the shapes of biological molecules and study how they physically interact with each other. This can provide insights that aren’t available through other methods and allow researchers to identify opportunities, for example, to block or enhance the activity of a particular protein or complex of proteins.

This new study provides definitive evidence about how these secondary DNA structures can pose physical barriers to DNA replication machinery, along with raising new questions about how problems might be resolved to allow replication to be completed.

“When our cells divide, our DNA needs to be copied so that a complete set of genetic instructions is passed down from a parent cell to new daughter cells,” Dr. Hite says. “The replication process is carried out by large protein complexes with multiple subunits, called replisomes. Replisomes orchestrate the unwinding of DNA before synthesis of new DNA to be distributed to the daughter cells.”

During cell division, the familiar double-stranded DNA helix gets split into two single strands, and the cell’s replication machinery moves along these single strands like a monorail, explains Allwein, a doctoral student at Weill Cornell Medicine.

“What these cryo-EM images showed us is that the G4 structure can get trapped—like an obstacle on the monorail track—inside the center of the ring-shaped protein complex called the CMG helicase that serves as the engine for unwinding the strands,” he says.

By uncovering precisely how G4s can block replication, scientists can now use that understanding to inform future studies and develop treatments that involve this critical cellular process.

“If these obstacles always led to an irreversible stall, we would never have successful cellular division,” Dr. Batra adds. “So this will also help us learn more about mechanisms by which DNA gets repaired, modified, and corrected during replication. Problems in these processes are associated with a number of diseases, including cancer and neurodegeneration.”

An unexpected discovery

The researchers made an additional, unexpected discovery about how the CMG helicase manages to travel along DNA strands.

“Proteins move along DNA strands to read and process genetic information all the time,” Dr. Remus says. “But in most cases, we still don’t understand what’s really happening at the molecular level. How do proteins actually—physically—move along DNA?”

It’s a challenging process to capture in action at the atomic scale needed to see what’s going on. Studies on bacteria and viruses have long provided a working model.

“Our study showed, however, that in complex organisms like people, this enzyme moves completely differently,” Dr. Hite adds.

In their paper, the scientists describe its movement as a “helical inchworm,” meaning that it shifts between two states—a flat and a spiral shape—as it encircles DNA strands.

“And the oscillation between these two states is what propels it along the DNA—allowing it to unwind those 3 billion base pairs each time the cell divides,” Dr. Hite says.