Enzymes and proteins work together to tidy up the ends of DNA in dividing cells

University of Wisconsin-Madison researchers have described how an enzyme and proteins interact to maintain the protective caps, called telomeres, at the ends of chromosomes, a new insight into how a human cell preserves the integrity of its DNA through repeated cell division.

DNA replication is essential to sustaining life as we know it, but many of the complexities of the process — how myriad biomolecules get where they need to go and interact through a series of intricately orchestrated steps — remain mysterious.

“The mechanisms behind how this enzyme called polα-primase works has been elusive for decades,” says Ci Ji Lim, assistant professor of biochemistry and principal investigator on new research on 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 about how Polα primase – a central cog in the DNA replication machine – works.”

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 nail protects the end of a shoelace. Eventually, telomeres are so short that vital genetic code is exposed on a chromosome and the cell, unable to function normally, enters a zombie state. Part of routine maintenance of a cell involves preventing excessive truncation by replenishing that DNA with 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). The scientists thought that Polα primase would have to change shape when switching from RNA to DNA molecule synthesis. Lim’s lab found that Polα-primase makes the RNA-DNA primer on telomeres using a rigid scaffold with the help of another cog in the telomere replication machine, an accessory protein called CST. CST acts like a stop-and-go sign, stopping the activity of other enzymes and bringing Polα-Primase to the job site.

“Prior to this study, we needed to visualize how Polα primase works to complete telomere replication at the ends of chromosomes,” says Lim. “Now we have high-resolution structures of Polα primase bound to an accessory protein complex called CST . We found that upon binding to the template DNA strand at the telomere, CST facilitated the action of Polα-primase. CST enables Polα primase to synthesize first RNA and then DNA using a unified architectural platform.”

The researchers also gained insight into how Polα primase might 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 repaired and where DNA replication has stalled.

“Since Polα primase plays a central and very important role in DNA replication in telomeres and elsewhere along the 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 Polα primase can also perform its task during genomic DNA replication,” says Lim complicated process to accomplish.”

“Our results demonstrate an unprecedented role that CST plays in facilitating this Polα primase activity,” explains first author Qixiang He, a graduate student in UW-Madison’s Biophysics graduate program. “It will be interesting to see if accessory factors involved in DNA replication elsewhere on chromosomes assemble polα-primase in the same way CST does for telomeres.”

Researchers built the structural model of the 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, resulting in high-resolution 3D models of biomolecules such as the enzymes that work in DNA replication.

Lim’s team used single particle cryo-EM analysis to first determine the structure of the CST polα primase and then focus on visualizing moving parts of the complex in more detail. They collected data at the UW-Madison Cryo-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 puzzle from our biochemical assay, but when we imaged the CST-Pol-α-primase co-complex and saw its cryo-EM structures, everything became immediately clear. This was extremely satisfying for everyone on the team. In addition, therefore, the structures also provide ideas that we can now test with experiments,” says Xiuhua Lin, laboratory director and co-author of the new study.

One of these ideas is to understand in more detail how CST-pol-α/primase works. The researchers also want to map the entire replication process of human telomeres and study how CST-pol-α/primase ceases its activity once the DNA has been copied to telomeres.

“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,” Lim says. “This is where the power and versatility of cryo-electron microscopy single-particle analysis comes into play. This approach allowed us to assemble a high-resolution atomic model and provided crucial insights into its motion, which in turn facilitated our understanding of how the human CST polα primase works.”