The Loader of the Rings in DNA

The DNA clamp loader positions the DNA polymerase machinery onto the DNA for replication. Chemistry professor John Kuriyan and his colleagues recently solved the structure of the yeast's complex. Image courtesy Gregory Bowman. Click on the image to see a larger version.

What do DNA replication and a new bottle of wine have in common? According to a new model proposed by chemistry professor John Kuriyan and his colleagues, they both employ a corkscrew motif. Based on the crystal structure of a donut-shaped ring complex that encircles DNA during replication, the new model helps to explain how DNA is snugly wrapped by the replication complex as DNA is duplicated.

The details of the structure and the model are reported in the June 17 edition of Nature by Kuriyan, postdoctoral fellow Gregory Bowman and Mike O'Donnell, who is at Rockefeller University.

At first glance, DNA replication seems simple: as the double helix is unwound into two single strands, the principle of complementarity dictates what base is added at each position of the two newly synthesized chains. However, the two template strands run in different directions, but DNA polymerase, the protein complex that copies DNA, works in only one direction. One strand, the leading strand, is copied continuously while the other, the lagging strand, is replicated in discontinuous loops, with each strand being handled simultaneously by a polymerase machine containing a dozen or more protein subunits.

The polymerases move remarkably fast, giving rise to many topological issues, said Kuriyan. "If you scale this process up to our level, it is comparable to a car moving at 350 mph while rotating quickly around an axis. In bacteria, the polymerase spins around the DNA at an amazing rate of 100 times per second."

One way the cell deals with all of this twisting and turning is to attach ring-shaped proteins, known as sliding clamps, to DNA. The DNA polymerase is connected to the sliding clamp, and the DNA is threaded through the ring. The polymerase can then detach from the DNA but remain tethered through the clamps.

"We found that subunits in this complex interact to give a symmetric spiral conformation with the same curvature as DNA," said Bowman. "The protein is poised to encircle DNA's corkscrew shape and load the clamp protein, which can slide down DNA and position the polymerase machinery."

This new crystal structure, the first from a eukaryotic source, included both the DNA clamp and ATP, the common energy currency of the cell. The clamp loader binds to the clamp and places it onto double-stranded DNA. "The two general functions of the clamp loader are to open, load and unload the clamp, and to target the clamp to the DNA as it becomes unwound for replication," said Kuriyan. With the bacterial clamp loader structure in hand, scientists knew what it looked like, but for loading to occur, ATP must be utilized.

This new structure also provided insight into how the ATPase activity of the clamp loader is triggered, leading to the loading of the clamp and the dissociation of the loader. "It was known that DNA binding triggers the ATPase of the clamp loader, but it was not clear how," said Bowman. "From the model, we see that the DNA itself, fitting cleanly inside the clamp loader, may very well trigger the activity directly."

The polymerase in charge of replicating the lagging strand must hop from one completed fragment to the next start site, called a primer-template junction. By specifically placing clamps onto these junctions, the clamp-loader effectively provides a landing pad that helps to target the polymerase for the next round of synthesis. How the clamp-loader recognized these junctions has been a mystery, but may very well be answered by the new structure. The primer-template junctions are short stretches of double-stranded DNA, somewhat like a stiff rod, that are flanked by the much more flexible single-stranded DNA. "The spiraling of the subunits forms a pocket on the underside of the clamp-loader which appears well suited to fit both the more rigid double helix, and a single-stranded extension," said Bowman. "In short, this pocket looks like an ideal binding site for a primer-template junction."

Kuriyan's group was the first to solve the structure of the ring-shaped clamp protein more than a decade ago, and in 2002 published the structure of the bacterial clamp-loader assembly, the five-subunit machine that loads the clamps onto DNA. This clamp-loader structure provided a good blueprint for the complex's architecture, but many details still needed to be filled in. "X-ray crystallography takes a snapshot of the protein complex and its components, a static picture of a dynamic process," said Bowman, "but with enough pictures, the movement and interactions in the complex can be seen." One analogy is a child's drawings of a dinosaur battle on corner of the pages in a book that, when flipped through quickly, becomes a rudimentary movie.

"The first structure was a picture of the complex in the 'off' state, and this is a snapshot of the activated state," added Bowman.

This structure is also the first from the AAA+ family to be solved in complex with a binding partner. The AAA+ superfamily proteins (ATPase Associated with various cellular Activities) have diverse cellular functions, including membrane fusion and protein degradation, but they share the ability to bind to their target and somehow change the structure. So far, the particular spiral seen in this structure is quite different than other AAA+ proteins, which have a more flattened disk-like shape. Thus, while other AAA+ proteins perform very different tasks in the cell, this X-ray snapshot of the clamp-loader may also indicate something about how this family of machines jostle about in their daily routines.