A protein thought to play a supporting role in DNA replication actually facilitates the whole process

Every time a cell divides, it must copy its entire genome so that each daughter cell inherits a complete set of DNA. During that process, enzymes known as polymerases race along the DNA to copy its code and build new strands. To prevent these machines from detaching mid-copy, a clamp-like protein tethers the polymerases to DNA, while another protein, Replication Factor C (RFC), snaps that ring into place. 

But new research demonstrates the RFC does much more than that. The findings, published in Cell, show RFC remains bound to the protein clamp even after loading it onto DNA and, together with a polymerase, the trio slides along the DNA as a unit, ensuring fast and reliable copying. The findings modify decades of textbook knowledge in basic biology and could help explain molecular roots of cancer and neurological disorders. 

"This study required us to combine biochemistry, single-molecule biophysics, and genetics," says Michael O'Donnell, head of the Laboratory of DNA Replication. "The new knowledge we gained as a result was beautiful."  

Maestro of the replication fork 

DNA replication requires polymerases that can faithfully copy the genome without falling off the DNA track, a property known as processivity. This stability is conferred by DNA sliding clamps—ring-shaped proteins that encircle double-stranded DNA and tether polymerases to their templates.  

In eukaryotes, the sliding clamp is proliferating cell nuclear antigen (PCNA), a molecule so central to replication that it is often described as the maestro of the replication fork. Because PCNA is a closed ring, however, it must be loaded onto DNA by a dedicated clamp loader, Replication Factor C, which opens the ring, positions it around DNA, and closes it again. For decades, RFC was believed to perform this one catalytic task before disengaging, leaving the clamp free to coordinate polymerases and other factors. 

Despite this long-held view, however, researchers on O’Donnell’s team recognized that the system might be more nuanced.  

It’s not unusual for his lab to spot something others missed. O'Donnell has spent decades pondering the replication fork—uncovering how clamps, clamp loaders, and helicases choreograph the duplication of the genome. His laboratory was the first to show that circular, sliding clamp proteins encircle DNA to hold polymerases in place, a concept that became a tenet of basic biology. "We discovered the sliding clamp protein thirty years ago, before any protein was known to slide on or encircle DNA," O'Donnell says. "Since that original finding, several repair and replication proteins have been found to function by encircling DNA”. 

Building on this foundation, his group went on to redefine the replisome, revealing with cryo-electron microscopy that the eukaryotic helicase engages DNA in the opposite orientation than previously assumed, showing biochemically how helicases melt DNA at origins. They also used single-molecule techniques to follow the gymnastics of the helicase and its precursor on DNA in a long-running collaboration with Shixin Liu’s Laboratory of Nanoscale Biophysics and Biochemistry. These discoveries reshaped our understanding of how replication begins, and why it remains stable.  

For O’Donnell, probing the mysteries of the replication fork was both a fresh scientific question and the continuation of a longstanding laboratory tradition. In collaboration with Liu’s team at Rockefeller and colleagues at Memorial Sloan Kettering, his group set out to clarify the true roles of RFC and PCNA. 

Load, stay, and synthesize 

The investigation began with a single DNA strand, stretched taut between two microscopic beads held in place by lasers, a device known as an optical trap. This setup allows the DNA to be manipulated like a tiny, tethered rope. Onto this DNA, the team added fluorescently labeled proteins: RFC glowing green and PCNA glowing red. Confocal microscopy then tracked the flashes of color as the proteins bound and moved. When the two proteins traveled together, they overlapped, their colors merging to yellow.  

"It's a powerful method, because you can correlate single-protein behavior and composition with changes in DNA mechanics in real time and at high resolution," says Gabriella Chua, a postdoctoral associate in O'Donnell's lab and a former graduate student in Liu's lab. "Because by nature, replication fundamentally alters DNA mechanics, this tool is very useful for studying this dynamic process." 

To determine what RFC’s lingering presence actually meant for DNA replication, the team then used biochemical techniques to test what happened when they introduced a mutant version of RFC that could not remain tethered. With normal RFC, the polymerase latched on and copied smoothly, staying in place until the job was done. But with the mutant clamp loader, the polymerase kept detaching from the clamp and slipped off the DNA, forcing repeated restarts and dramatically slowing the reaction. 

"It's not a result that a lot of people were expecting,” Chua says. “RFC was thought to do its job and leave the scene. But in hindsight it makes total sense given the recent results from other labs that hinted that RFC might function more than a catalytic loader.” 

Genetic experiments performed by the laboratory of Xiaolan Zhao, who did postdoctoral work at Rockefeller with the late Günter Blobel and now heads her own’s lab at Memorial Sloan Kettering, backed up the biochemical and single-molecule findings. In yeast, the researchers engineered a version of RFC that was weakened in its ability to stabilize the PCNA-polymerase complex. On its own, this mutant strain could still grow, but when the same cells were also missing another enzyme FEN1, they could not survive. In the context of the new model, the result demonstrates that RFC's architectural role is so essential that the system contains built-in redundancies. Besides its own enzymatic function, FEN1 also harbors a structural role that can stand in for RFC to keep the replication machinery moving. 

The PCNA clamp has also emerged as a potential cancer drug target, because cancer cells depend so heavily on its recruiting capacity to proliferate. PCNA serves as a central hub that binds dozens of different proteins involved in replication and repair through distinct sites, and its interactions could be blocked selectively. For example, a drug designed to interfere only with RFC and FEN1 interactions—while still allowing polymerase binding—could disrupt tumor growth without halting replication entirely.   

The study also has implications for a broader, emerging theme in biology: proteins often carry out important non-catalytic functions, in structure or scaffolding, that extend well beyond their known enzymatic activities.  

"Our study shows that we can discover new roles for proteins even in a well-studied system like DNA replication," Chua says. "Non-catalytic functions for proteins are easy to overlook but serve crucial roles in biology. We should be searching for them more often."