Mechanism that unwinds DNA may function similar to an oil rig 'pumpjack'

"DNA replication is a major source of errors that can lead to cancer," explained Li, a Professor in the Department of Biochemistry & Cell Biology at Stony Brook University, a scientist at Brookhaven Lab, and lead author of the paper. "The entire genome -- all 46 chromosomes -- gets replicated every few hours in dividing human cells," Li said, "so studying the details of how this process works may help us understand how errors occur."

The investigative team includes scientists from Stony Brook University, the U.S. Department of Energy's Brookhaven National Laboratory, Rockefeller University, and the University of Texas. Their research builds on previous collaborative work led by Dr. Li. In 2015, they produced the first-ever images of the complete DNA-copying protein complex, called the replisome. That study revealed a surprise about the location of the DNA-copying enzymes -- DNA polymerases.

In the new paper, titled "Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation," the research team focused on the atomic-level details of the "helicase" portion of the protein complex -- the part that encircles and splits the DNA double helix so the polymerases can synthesize two daughter strands by copying from the two separated parental strands of the "twisted ladder."

The scientists produced high-resolution images of the helicase using a technique known as cryo-electron microscopy (cryo-EM). One advantage of this method is that the proteins can be studied in solution, which is how they exist in the cells.

"You don't have to produce crystals that would lock the proteins in one position," Li said, adding that this is essential because the helicase is a molecular "machine" made of 11 connected proteins that must be flexible to work. "You have to be able to see how the molecule moves to understand its function."

"The whole mechanism operates similar to an old style pumpjack oil rig, with one part of the protein complex forming a stable platform, and another part rocking back and forth," Li explained. "Each rocking motion could nudge the DNA strands apart and move the helicase along the double helix in a linear fashion," he suggested.

Using computer software to sort out the images revealed that the helicase has two distinct conformations -- one with components stacked in a compact way, and one where part of the structure is tilted relative to a more "fixed" base.

The atomic-level view allowed the scientists to map out the locations of the individual amino acids that make up the helicase complex in each conformation. Then, combining those maps with existing biochemical knowledge, they came up with a mechanism for how the helicase works.

"One part binds and releases energy from a molecule called ATP. It converts the chemical energy into a mechanical force that changes the shape of the helicase," Li said. After kicking out the spent ATP, the helicase complex goes back to its original shape so a new ATP molecule can come in and start the process again.

"It looks and operates similar to an old style pumpjack oil rig, with one part of the protein complex forming a stable platform, and another part rocking back and forth," Li said. Each rocking motion could nudge the DNA strands apart and move the helicase along the double helix in a linear fashion, he suggested.

This linear translocation mechanism appears to be quite different from the way helicases are thought to operate in more primitive organisms such as bacteria, where the entire complex is believed to rotate around the DNA, Li said. But there is some biochemical evidence to support the idea of linear motion, including the fact that the helicase can still function even when the ATP hydrolysis activity of some, but not all, of the components is knocked out by mutation.

"We acknowledge that this proposal may be controversial and it is not really proven at this point, but the structure gives an indication of how this protein complex works and we are trying to make sense of it," he said.