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Researchers from the University of Wisconsin-Madison have described how an enzyme and proteins interact to maintain protective caps, called telomeres, at the end of chromosomes, a new insight into how a human cell maintains the integrity of its DNA by repeated cell division.
DNA replication is essential for perpetuating life as we know it, but many of the complexities of the process – how myriad biomolecules get to where they need to go and interact in a series of orchestrated steps of complex way – remain mysterious.
“The mechanisms underlying the functioning of this enzyme, called Polα-primase, have been elusive for decades,” says Ci Ji Lim, assistant professor of biochemistry and principal investigator on new DNA replication research published recently. in Nature. “Our study constitutes a major advance in the understanding of DNA synthesis at the ends of chromosomes and generates new hypotheses on the functioning of Polα-primase – a central cog in the DNA replication machinery.”
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 a needle protects the end of a shoelace. Eventually, the telomeres are so short that a chromosome’s vital genetic code is exposed and the cell, unable to function normally, enters a zombie state. Part of a cell’s routine maintenance includes preventing excessive shortening by replenishing this DNA using Polα-primase.
At the telomere construction site, Polα-primase first builds a short nucleic acid primer (called RNA) and then extends this primer with DNA (then called RNA-DNA primer). Scientists thought that Polα-primase should change its shape when it goes from synthesizing RNA to synthesizing DNA molecules. Lim’s lab discovered that Polα-primase makes the RNA-DNA primer at telomeres using a rigid scaffold using another cog in the telomere replication machinery, an accessory protein called CST. CST acts as a stop-and-go sign that shuts down 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,” Lim says. “Now we have high-resolution structures of Polα-primase bound to an accessory protein complex called CST. We found that after CST binds to the template DNA strand at the telomere, it facilitates the action of Polα-primase. By doing so, CST sets the stage for Polα-primase to first synthesize RNA and then DNA using a unified architectural platform.
The researchers also got a glimpse of how Polα-primase could initiate DNA synthesis elsewhere along the length of a chromosome. Other scientists have also found the CST-pol-α-primase complex at sites where DNA damage is being repaired and DNA replication is blocked.
“Because Polα-primase plays a central and very important role in DNA replication in telomeres and elsewhere along chromosomes – it is the only enzyme that makes primers on DNA templates from zero for DNA replication – our CST-Polα-primase structure provides new insights into how polα-primase can also do its job during genomic DNA replication,” Lim explains. is a very elegant solution that nature has developed to accomplish this complicated process.”
“Our results reveal an unprecedented role that CST plays in facilitating this Polα-primase activity,” says 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 chromosomes configure Polα-primase in the same way that CST does for telomeres.”
The researchers constructed the structural model of CST-Polα-primase 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 and then imaged with a transmission electron microscope, yielding high-resolution 3D models of biomolecules like enzymes involved in the replication of DNA.
Lim’s team used cryo-EM single-particle analysis to first determine the structure of CST-Polα-primase and then focus on more detailed visualization of the moving parts of the complex. They collected data at the UW-Madison Electron Microscopy Research Center (CEMRC), housed in 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 riddle from our biochemical assay, but once we imaged the CST-pol-α-primase co-complex and saw its cryo-EM structures, everything immediately became clear. It was extremely satisfying for everyone on the team. Beyond that, the structures also provide insights that we can now design experiments to test,” says Xiuhua Lin, lab manager and co-author of the new study.
Among these ideas is the more detailed functioning of CST–pol-α/primase. The researchers also want to map the entire process of human telomere replication and study how CST–pol-α/primase terminates its activity once telomere DNA is copied.
“You can’t really study how a car moves by looking at its individual parts — you have to put the parts together and see how they work together. But biomolecular machines often have so many moving parts that they can be difficult to study,” says Lim. “This is where the power and versatility of single-particle analysis by cryo-electron microscopy comes in. This approach allowed us to create a high-resolution atomic model and provided critical information about how it moves. , which in turn has facilitated our understanding of how human CST-Polα-primase works.