Tim Grant Tim Grant

Cryo-EM studies reveal the ‘high-wire act’ of bacterial replication

Bacteria have a replication problem. In nearly every attempt to copy their genome, some kind of glitch — it could be exposure to UV light, or an obstruction within the cell — occurs that throws the process off.

Normally this would be a lethal error, but bacteria have finely-tuned proteins that race to the site of the break and repair it on the fly. This “replication restart” process is a little-understood but essential activity for bacteria — and thus, a very intriguing future target for antibiotic development.

James Keck, a professor of biomolecular chemistry at UW–Madison, has been studying the replication restart process for years, and has used crystallography techniques to determine the structures of key proteins that guide the DNA repair. But the replication restart process is so dynamic, Keck says, these structures rarely “sit tight” long enough to get detailed images through crystallography.

Keck has taken a huge leap in this inquiry through a partnership with Tim Grant, a Morgridge Institute for Research investigator, UW–Madison biochemistry professor and pioneer in cryo-electron microscopy (cryo-EM).  In a study published in May in the journal Nature Communications, the team describes using cryo-EM to reveal, for the first time, a switch-like mechanism that initiates the restart process and a major restructuring of proteins that allows DNA repair to take hold.

“We were able to look at the structure of one protein, called PriA, that serves as a first responder and senses abandoned replication processes,” Keck says. “We had pictures of PriA before, but we’ve seen now in cryo-EM just how radically the structure changes when it recognizes DNA. If you were to inactivate that protein in some bacterial pathogens, it would be enough to kill the bacteria.”

They also were able to get a better view of the overall repair process, which involves three distinct proteins in their model E. coli. Their imaging revealed how these repair proteins open up a pore to interact directly with the DNA strands, while exposing protein surfaces that trigger replication restart.

bacterial protein PriA
This GIF, developed from cryo-EM data, illustrates how the bacterial protein PriA undergoes a dramatic unfolding, allowing it to envelop and fix broken strands of DNA.

Grant used a software package he developed called cisTEM to process the cryo-EM data, which was drawn from more than 2,700 movies. The software provides a more user-friendly interface for biologists and is adept at analyzing and predicting motion in biological processes. Data was collected at the UW–Madison Cryo-EM Research Center.    

“Cryo-EM showed us that a large motion in one protein is key to controlling the process,” Grant says. “It achieves a number of cool things, opening up a pore that encircles one part of the DNA and opening up interfaces for other proteins. The process cannot continue until this movement occurs.”

Chromosomes in bacteria are circles of DNA that duplicate starting at a single origin point, with two replication processes running around the circle bidirectionally until they meet at the bottom — basically, two events starting at 12 o’clock and meeting together at 6 o’clock. This differs from the linear replication of DNA in human cells. For bacteria, replication mistakes can happen anywhere along that clock face, a big reason why the replication restart process is so critical to survival.

“That makes replication a little bit of a high wire act for bacteria,” Keck says.

Despite the differences between bacteria and eukaryotic cells, having a basic understanding of the bacterial replication restart machinery will have benefits for biomedical pursuits beyond possible antibiotic development, Keck says. Human DNA repair systems are studied extensively because they’re so important to understand with respect to cancer and chemotherapy. The widely used chemotherapy drug Cisplatin, for example, works by actively knocking down the capacity of tumors to replicate and repair DNA.

“Our findings give us more clues about what kind of features we might expect in a protein that does this kind of thing in humans,” Keck says. “And that helps you narrow the list of potential candidates in the human proteome to perhaps a handful that warrant further study.”