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August 27, 2004
DNA's Grand Prix

Imagine a car racing along a track at 300 miles per hour. Now imagine the car spinning and spiraling as it careens, each blurred movement perfectly coordinated.

Such are the acrobatics performed by protein structures during DNA replication. Hoping to shed light on this poorly understood process, a team led by John Kuriyan of Berkeley Lab's Physical Biosciences Division has captured a split-second glimpse of a speedy protein complex that plays an essential role in DNA's ability to make copies of itself.

The clamp-loader (blue) bound to the clamp (gold ring), with double-stranded DNA modeled through the clamp and against the underside of the clamp-loader.

Their work, reported in Nature, reveals for the first time how a ring-shaped protein called a sliding clamp may target a special DNA structure generated during replication, in which a polymerase enzyme adds free nucleotides to a single DNA strand. As part of this process, the polymerase uses the sliding clamp like a seatbelt to tether itself to the new DNA double helix.

This protein assembly churns through 1,000 base pairs per second, moving 30 times its length every second. At a human scale, that's the equivalent of racing at almost one-half the speed of sound. But the protein complex doesn't just race. It also rotates 100 times per second as it follows DNA's spirals. And further complicating matters, the polymerase moves in only one direction while the two DNA strands to be replicated are arranged in opposite directions. This means that while one DNA strand can be smoothly replicated by the polymerase, the other strand can only be duplicated in many shorter stretches, with a second polymerase hopping from one piece to another, and its sliding clamp continuously clamping and unclamping from DNA like an automated claw on an assembly line.

"The complexity of its speed and movement is mind-boggling," says Kuriyan, who is also a Howard Hughes Medical Institute investigator and a Chancellor's Professor in the University of California at Berkeley's Department of Molecular and Cell Biology and Department of Chemistry. He conducted the research with fellow Physical Biosciences Division researcher Gregory Bowman and Rockefeller University's Mike O'Donnell. "But nature is very smart and its solutions are tremendously simple."

To determine how the sliding clamp targets DNA that is ready to replicate itself, encircles it, then lets go when the replication is complete — all in the blink of an eye — Kuriyan's group turned to Berkeley Lab's Advanced Light Source. There, with the help of Physical Biosciences' Corie Ralston and Gerry McDermott, they used x-ray diffraction to reveal the structure of a sliding clamp from a yeast species as it's bound to a clamp loader, a five-subunit protein motor that both opens and closes the clamp, and targets it toward freshly unwound DNA strands that are ready for replication. The researchers were able to capture this protein assembly precisely when the clamp loader is poised to release the sliding clamp around its target DNA.

The result is a three-dimensional protein structure that reveals how the clamp loader simultaneously binds to the clamp and ATP, a high-energy molecule that fuels the motor. And from this structure, coupled with a battery of experiments from postdoctoral fellows Marjeta Podobnik, Steven Kazmirski and Eric Goedken, Kuriyan's group can begin to understand how the protein assembly recognizes DNA strands that are ready for replication — a fundamental process that is conserved in every branch of life.

"The clamp loader recognizes the junction where DNA changes from double strand to single strand," says Kuriyan. "And it couples this recognition with a structure that forces the clamp loader to release the clamp around DNA."

This work marks yet another research milestone in Kuriyan's group, which was the first to solve the structure of the clamp protein more than a decade ago. Next, to gain a more global understanding of how clamp loading works, Kuriyan hopes to determine how the entire protein assembly is configured on DNA.

"Our ultimate goal is to determine the structure of the entire replication assembly at this level of atomic detail," says Kuriyan. "While it may take us a while to accomplish that task, we are excited about this first structure of a DNA-clamp loader complex in action, which we resolved using x-ray crystallography."

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