Enzymes, proteins work together to tidy up the tail end of DNA in dividing cells

Researchers at the University of Wisconsin-Madison have described the way enzymes and proteins interact to maintain protective coverings, called telomeres, at the ends of chromosomes, a new insight into how human cells maintain the integrity of their DNA through repeated cell division. .

DNA replication is critical to sustaining life as we know it, but much of the complexity of the process — how myriad biomolecules get to where they need to be and interact through a series of intricately orchestrated steps — remains a mystery.

From left, Qixiang He, Ci Ji Lim, Xiuhua Lin.

“The mechanism behind how this enzyme, called Polα-primase, works has been elusive for decades,” said Ci Ji Lim, assistant professor of biochemistry and principal investigator on a new study on DNA replication published recently in Nature. “Our study provides a major breakthrough in understanding DNA synthesis at the ends of chromosomes, and generates new hypotheses about how Polα-primase – the main cog in the DNA replication machinery – operates.”

Each time a cell divides, the telomeres at the ends of the long DNA molecules that make up one chromosome shorten slightly. Telomeres protect chromosomes like aglets protect the ends of shoelaces. Eventually, the telomeres become so short that the vital genetic code on the chromosomes is exposed and the cell, which cannot function normally, enters a zombie state. Part of routine maintenance of cells includes preventing excessive shortening by refilling this DNA using Polα-primase.

At the telomere construction site, Polα-primase first builds a short nucleic acid primer (called RNA) and then expands this primer with DNA (later called RNA-DNA primer). Scientists think Polα-primase needs to change its shape when it goes from RNA to DNA molecule synthesis. Lim’s laboratory found that Polα-primase makes RNA-DNA primers in telomeres using a rigid scaffold with the help of another cog in the telomere replication machinery, an accessory protein called CST. CST acts like a stop-and-go signal that stops the activity of other enzymes and brings Polα-primase to the construction site.

“Before this study, we had to imagine how Polα-primase works to complete telomere replication at the ends of chromosomes,” said Lim. “Now, we have a high-resolution structure of Polα-primase bound to an accessory protein complex called CST. We found that once CST binds to the template DNA strand in telomeres, it facilitates the action of Polα-primase. By doing so, CST set the stage for Polα-primase to synthesize RNA first and then DNA using a unified architectural platform. ”

The researchers also glimpsed how Polα-primase could initiate DNA synthesis elsewhere along the chromosome. Other scientists have also found CST-pol-α-primase complexes at sites where DNA damage is being repaired and where DNA replication stops.

“Because Polα-primase plays a central and very important role in DNA replication in telomeres and elsewhere along chromosomes — it’s the only enzyme that makes primers on DNA templates from scratch for DNA replication — the structure of our CST-Polα-primase provides insight new research on how Polα-primase can also do its job during genomic DNA replication,” said Lim. “This is a very elegant solution that nature has evolved to complete this complex process.”

“Our findings reveal the unprecedented role that CST plays in facilitating this Polα-primase activity,” explains first author Qixiang He, a graduate student in the UW–Madison biophysics graduate program. “It will be interesting to see if accessory factors involved in DNA replication elsewhere on the chromosome regulate Polα-primase in the same way that CST does for telomeres.”

The researchers built a structural model of CST-Polα-primase using an advanced imaging technique called single-particle analysis cryo-electron microscopy. In cryo-EM, the frozen sample is rapidly suspended in a thin layer of ice, then imaged with a transmission electron microscope, generating high-resolution 3D models of biomolecules such as enzymes working in DNA replication.

Lim’s team used cryo-EM single-particle analysis to first determine the structure of CST-Polα-primase and then visualize the moving parts of the complex in more detail. They collected data at the UW–Madison Cryo-Electron Microscopy Research Center (CEMRC), housed at the UW–Madison Department of Biochemistry, and the NCI-funded National Cryo-Electron Microscopy Facility at the Frederick National Laboratory for Cancer Research.

“We started with a puzzle from our biochemical assay, but once we imaged the CST-pol-α-primase co-complex and looked at its cryo-EM structure, everything immediately became clear. This is very satisfying for everyone on the team. Beyond that, the structure also provides the idea that we can now design experiments to test,” said Xiuhua Lin, lab manager and co-author of the new study.

Among these ideas is capturing how CST–pol-α/primase works in greater detail. The researchers also wanted to map the entire process of human telomere replication, and they studied how CST–pol-α/primase stops its activity after the DNA in the telomeres is copied.

“You can’t really learn how a car moves by looking at the individual parts — you have to assemble the parts and observe how they work together. But biomolecular machines often have so many moving parts that they are difficult to study,” said Lim. “That’s where the power and versatility of cryo-electron microscopic single-particle analysis comes in. This approach allowed us to construct high-resolution models of the atom and provided critical insight into how it moves, which in turn facilitated our understanding of how human CST-Polα-primase works.”

It this study was supported by a grant from the National Institutes of Health (R00GM131023).

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