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August 5, 2004

 

 

Structure of a DNA Clamp–Loader complex

Sliding clamps are ring-shaped proteins that encircle DNA and enable polymerases—enzymes that replicate DNA—to relax and regain their hold on DNA strands without losing their place, despite the considerable torque that results from the production of double-helical DNA. In addition to their role in DNA replication, sliding clamps are also involved in several other processes that require a mobile contact on DNA. Hoping to shed light on this important but still poorly understood mechanism, a trio of researchers from Berkeley and New York have obtained the crystal structure of a sliding clamp in complex with a clamp loader assembly that is "powered" by the hydrolysis of ATP. The researchers found a spiral structure in the clamp loader with a striking correspondence to the grooves of the DNA double helix. The crystal structure suggests a simple explanation for how this interaction with the double helix triggers ATP hydrolysis and the release of the sliding clamp.

Clamp loading cycle
Schematic of the clamp loading cycle. PCNA is the clamp, RFC is the clamp loader. Hydrolysis of ATP releases the loader from the clamp.

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. Kuriyan et al. have captured a split-second glimpse of a speedy protein complex that plays an essential role in DNA's ability to make copies of itself. Their work 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.

The clamp–loader protein assembly churns through 1000 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, 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—the research group turned to ALS Beamline 8.2.2. There, they used x-ray diffraction to reveal the structure of a sliding clamp from a yeast species as it is 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. From this structure, scientists 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 stranded to single stranded, and it couples this recognition with a structure that forces the clamp loader to release the clamp from around the DNA.

This work marks yet another milestone for this research 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, the scientists hope to determine how the entire protein assembly is configured on DNA. The group's ultimate goal is to determine the structure of the entire replication assembly at this level of atomic detail. While it may take a while to accomplish that task, the resolution of this first structure of a DNA clamp–loader complex in action using x-ray crystallography is an exciting step in the right direction.

Clamp–loader complex

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

Research conducted by G.D. Bowman and J. Kuriyan (University of California, Berkeley, and Berkeley Lab) and M. O'Donnell (The Rockefeller University).

Research funding: National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: G.D. Bowman, M. O'Donnell, and J. Kuriyan, "Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex," Nature 429, 724 (2004).

ALSNews Vol. 243, July 28, 2004

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