If the engines of DNA replication stay in high gear all the time, they eventually stall or even break down. Fortunately, these engines, which consist of protein complexes, have what could be called idle modes, or proteostasis mechanisms. One such mechanism—for pausing or stopping the replication of DNA’s “leading strand”—has already been up on the lift, so to speak. Its parts are known. But what about the proteostasis mechanism for pausing or stopping the replication of DNA’s “lagging strand”?

As it happens, the mechanism for the lagging strand was just taken in for inspection. And the inspection, which was performed by scientists at the University of Pennsylvania and University of Leeds, not only yielded a parts list, but it also offered maintenance tips. Specifically, it suggested that adjustments to a faulty mechanism could help treat neurologic and developmental disorders.

Details about the inspection appeared in the journal Cell, in an article titled, “The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.” The article described how the scientists used cryo-electron microscopy, CRISPR-based mutation analyses, and other advanced techniques to identify a protein complex that has a central replication-stopping role for the lagging strand.

This four-protein machine, which is called 55LCC, binds to DNA and its associated replication complex. Powered by two ATPases, 55LCC appears to unfold the tightly folded replication complex, allowing it to be chopped up by protein-snipping enzymes and cleared away.

“Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability,” the article’s authors wrote. “55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.”

The DNA replication process is carried out by multiple protein complexes with highly specialized functions, including the unwinding of DNA and the copying of the two unwound DNA strands. The process is akin to a factory assembly line where balls made up of massive, crumpled strings of data are unraveled, allowing specific pieces to be trimmed and copied.

“We’ve found what appears to be a critical quality-control mechanism in cells,” said senior co-corresponding author Roger Greenberg MD, PhD, professor of cancer biology, director of the Penn Center for Genome Integrity, and director of basic science at the Basser Center for BRCA at Penn Medicine. “Trillions of cells in our body divide every single day, and this requires accurate replication of our genomes. Our work describes a new mechanism that regulates protein stability in replicating DNA. We now know a bit more about an important step in this complex biological process.”

The scientists found that stopping or pausing function of 55LCC is crucial for the smooth progression of DNA replication. When 55LCC is absent, replication is likely to become stuck, and affected cells cease dividing. “We eventually see massive changes to genome stability in these cells,” Greenberg noted. “Their chromosomes fail to segregate properly during cell division.”

The scientists suspect that 55LCC may be involved in regulating not just the DNA replication process associated with cell division, but also when DNA-damaging lesions block replication.

Inherited mutations in enzymes that help make up 55LCC are known to be associated with childhood syndromes involving hearing loss, cognitive and movement impairments, and epilepsy. The scientists showed in their experiments that these disease-causing mutations tend to reduce the structural stability of 55LCC or affect its interactions with other proteins.

“This work hopefully marks the start of a deeper understanding of these severe neurodevelopmental syndromes,” Greenberg remarked. “Ultimately, the implications of this finding could be much broader. It could lead to ways to mitigate the clinical issues associated with syndromes stemming from 55LCC dysfunction, which include epilepsy, hearing loss, mental retardation, and bone marrow insufficiency.”

55LCC may also turn out to be a more general tool for protein recycling—another process critical to the health of cells. Greenberg and his team are continuing to study how 55LCC works and is regulated, including understanding the precise signal that tells 55LCC to become active and start unfolding a DNA replication complex.

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