Researchers at the University of Wisconsin-Madison describe how an enzyme and protein interact to maintain protective caps called telomeres at the ends of chromosomes, a new insight into how human cells go through repeated cell divisions. How does it maintain the integrity of its DNA? ,
As we know, DNA replication is essential to sustain life, but the process’s many complications – how the myriad biomolecules get where they need to go and interact over a series of intricately arranged steps. – Remains mysterious.
“The mechanism behind how this enzyme, called polka-primase, works has been elusive for decades,” says C G Lim, assistant professor of biochemistry and principal investigator on the new research in DNA replication recently published in Nature. “Our study provides a major breakthrough in understanding DNA synthesis at the ends of chromosomes, and generates new hypotheses as to how a central cog in the DNA replication machine — the Pola-primase — operates.”
Each time a cell divides, the telomeres at the end of the long DNA molecule that makes up a single chromosome shorten slightly. Telomeres protect chromosomes like an aglet protects the end of a shovel. Eventually, telomeres are so short that the important genetic code on a chromosome is exposed and the cell, unable to function normally, enters a zombie state. Part of the routine maintenance of the cell involves preventing excessive shortening by replenishing this DNA using Polα-primase.
At the telomere construction site, Polα-primase first creates a short nucleic acid primer (called RNA) and then extends this primer with DNA (then called RNA-DNA primer). The scientists thought that Pola-primase would need to change its shape when it switched from RNA to DNA molecule synthesis. Lim’s lab found that Pola-primase cleaves RNA-DNA primers at telomeres using a rigid scaffold with the help of another cog in the telomere replication machine, a helper protein called Cst. The CST acts as a stop-and-go signal that inhibits the activity of other enzymes and brings Pola-primase to the manufacturing site.
“Prior to this study, we had to visualize how Pola-primase works to complete telomere replication at the ends of chromosomes,” says Lim. “Now, we have high-resolution structures of Polα-primase that bind to a helper protein complex called Cst. We found that after Cst binds to the template of the DNA strand at the telomere, it facilitates the action of Polα-primase In doing so, CST sets the stage for Pola-primase to synthesize first RNA and then DNA using an integrated architectural platform.
The researchers also got a glimpse of how Pola-primase could initiate DNA synthesis elsewhere along the length of the chromosome. Other scientists have also found Cst-Pol-α-primase complexes at sites where DNA damage is being repaired and where DNA replication has stopped.
“Since Polα-primase plays a central and very important role in DNA replication at telomeres and elsewhere along chromosomes – it is the only enzyme that creates primers on DNA templates from scratch for DNA replication – our CST-Polα-primase structure provides new insights into how Pola-primase may also perform its function during genomic DNA replication,” says Lim. “It’s a very beautiful solution that nature has developed to accomplish this complex process.”
“Our findings reveal an unprecedented role that CST plays in facilitating this Pola-primase activity,” explains first author Qixiang He, a graduate student in the UW-Madison Biophysics graduate program. “It will be interesting to see whether co-factors involved in DNA replication elsewhere on the chromosomes set up Pola-primase in the same way that CST does for telomeres.”
The researchers constructed a structural model of the Cst-Polα-prime using an advanced imaging technique called cryo-electron microscopy single-particle analysis. In cryo-EM, rapidly-frozen samples are suspended in a thin film of ice, then imaged with a transmission electron microscope, resulting in high-resolution, high-resolution images of enzymes working in DNA replication such as biofilms. – Contains 3D models of molecules.
Lim’s team used cryo-EM single particle analysis to first determine the structure of the Cst-Polα-prime and then house the moving parts of the complex in more detail. They collected data at the UW-Madison Cryo-Electron Microscopy Research Center (CEMRC), located in the UW-Madison Department of Biochemistry, and the NCI-funded National Cryo-Electron Microscopy Facility at 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 structures, everything became immediately clear. It was extremely satisfying for everyone on the team. Furthermore, the structures also provide ideas that we can now design experiments for testing,” says Xiuhua Lin, the laboratory manager and co-author of the new study.
These ideas are captured in more detail on how cst-pol-α/primage works. The researchers also want to map the entire human telomere replication process, and they are studying how cst-pol-α/primase ceases its activity once DNA is copied at telomeres.
“You can’t really study how a car moves by looking at its individual parts—you have to assemble the parts and observe how they work together. But biomolecular machinery often has so many moving parts.” It can be difficult to study,” Lim says. “This is where the power and versatility of cryo-electron microscopy single-particle analysis comes in. This approach allowed us to put together a high-resolution nuclear model and provided important insight into how it operates, which in turn facilitated our understanding of how the human CST-Polα-primase works. “
Reference: He Q, Lin X, Chavez BL, Agarwal S, Lusk BL, Lim CJ. Structures of the human CST–Polα–primase complex bound to the telomere template. Nature, 2022: 1-2. doi: 10.1038/s41586-022-05040-1
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