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Structures of Clamp-Loader Complexes Are Key to DNA Replication Print

DNA Replication:
An Open-and-Shut Case

Every time a cell divides, whether in humans or in other organisms, its chromosomes must be copied quickly but without mistakes. When copying errors do occur, the resulting mutations can lead to cancer or other life-threatening diseases, so understanding the copying process is important for improving human health. The protein that copies DNA (DNA polymerase) requires a ring-shaped protein complex, called the sliding clamp, to hold it onto the DNA, so that the polymerase can move at high speed. As it sequentially copies the nucleotides that make up the DNA strand, synthesis can occur as fast as 1000 nucleotides per second. However, the sliding clamp cannot get onto DNA by itself and requires a separate complex of proteins, called the clamp loader, to wrap the sliding clamp ring around DNA.

Recent research by Kelch et al. provides snapshots of clamp loaders in action. Their work clarifies the mechanism of DNA replication on a very detailed level, showing the many proteins involved in the process and how they interact synergetically to physically move along the DNA strand. For example, it shows how clamp loaders first break open the ring, so that DNA can slip into the central pore of the sliding clamp ring. Once DNA is bound, a switch flips in the clamp loader to close and release the sliding clamp around DNA, so that a polymerase can bind the clamp and start copying DNA.

DNA replication occurs when the enzyme DNA polymerase moves along DNA strands at high speed, copying nucleotides as it goes. A separate ring-shaped protein complex, called the sliding clamp, attaches the polymerase to the DNA with the help of a molecular machine, the clamp loader, whose action depends on ATP. How the clamp loader accomplishes this task was unknown until researchers from University of California, Berkeley, and Rockefeller University solved structures of the clamp loader bound to the sliding clamp, DNA, and an ATP analog. The structures, obtained at ALS Beamlines 8.2.1 and 8.2.2, reveal key insights into the mechanism by which the sliding clamp that facilitates replication of chromosomes is loaded onto DNA.

DNA replication is the most crucial step in cellular division, a process necessary for life, and errors can cause cancer and many other diseases. High-speed replication of chromosomal DNA (up to 1000 nucleotides per second) requires the DNA polymerase to be attached to a sliding clamp that prevents the polymerase from diffusing away from the DNA when it releases the DNA substrate during synthesis. In forms of all cellular life, sliding clamps are protein complexes that form rings around DNA, thereby providing a topological link for DNA polymerases to the DNA. Sliding clamps are also crucial components of various other cellular pathways, such as DNA repair, cell cycle control, and chromatin structure.

Sliding DNA clamps are loaded onto DNA by pentameric clamp-loader complexes. Among the two strands of the separated DNA (leading and lagging strands), the lagging-strand replication is semi-discontinuous; that is, it breaks into a chain of fragments (the Okazaki fragments). Clamp loaders must place a clamp at the start of each of these fragments in order to accomplish lagging-strand synthesis. Thus, the clamp loader is a crucial aspect of the DNA replication machinery. But the ring shape of the sliding clamp presents a topological problem: How is a closed circle loaded onto a chromosome?

Here is where the ATP comes in. Clamp loaders belong to the AAA+ family of ATPases, an important family of molecular enzymes that convert the chemical energy of ATP to mechanical work. When clamp loaders are in the ATP-bound state, they can bind with high affinity to the sliding clamp and, importantly, break the ring open and hold it in an open state. The open clamp/clamp-loader complex can then bind to so-called primer-template DNA to start the replication. Binding of DNA triggers the activation of the ATPase active sites. The subsequent hydrolysis of ATP causes the clamp loader to release from the clamp, which closes around the DNA. However, how clamp loaders accomplish these tasks was not known at the structural level.

To address the mechanism of clamp loading, the researchers solved the structure of the ternary complex of the clamp loader from bacteriophage T4 bound to a sliding clamp and DNA. The structure revealed that clamp-loader AAA+ modules form an ATP-dependent, right-handed spiral that matches the helical symmetry of DNA. The AAA+ spiral, in turn, holds the clamp in a right-handed, open, lock-washer shape, causing the clamp to be broken at one of the subunit interfaces.

Top: The structure of the clamp loader bound to an open sliding clamp and primer template DNA. The clamp loader comprises five subunits (A through E), each consisting of an AAA+ module and a collar region. The collar domains assemble into a circular cap. The AAA+ modules form a symmetric, right-handed spiral that wraps around the DNA and holds the sliding clamp into an open lock-washer shape. Bottom: The conformation of the open sliding clamp. The clamp is opened by ~ 9 Å. It consists of six domains, which are distorted from the closed, planar conformation by the clamp loader. The relative domain rotations are mapped onto the clamp (right).


Further, the researchers identified a mechanism occuring away from the ATPase active site (allosteric mechanism) for activation of the ATPases in response to DNA binding and also defined a conformational change that occurs in response to ATP hydrolysis. Hydrolysis of ATP begins from an end of the AAA+ spiral, which causes the symmetric AAA+ spiral to break down. This allows the clamp to close around DNA and causes the clamp loader to lose its symmetric recognition of the clamp and DNA, resulting in an elegant mechanism for ejection of the clamp loader. Thus the ATP-dependent spiral of AAA+ modules is the key for controlling the clamp loader's function.


Left: Structure of the clamp loader fully loaded with ATP and bound to an open clamp. The schematic at the bottom illustrates the clamp and the AAA+ modules of the clamp loader from the side such that all subunits can be seen simultaneously. In the ATP-loaded state, all AAA+ modules are positioned perfectly to match the clamp binding sites. Right: Structure of the clamp loader mimicking a post ATP-hydrolysis state in the B subunit and bound to a closed clamp. ATP hydrolysis causes the B subunit to disengage from the symmetric AAA+ spiral and from its binding site in the clamp, allowing the closure of the ring.


A detailed mechanism for the clamp loading reaction. (1) In the absence of ATP, the clamp loader AAA+ modules cannot organize into a spiral shape. (2) Upon ATP binding, the AAA+ modules form a spiral that can bind and open the clamp. (3) Primer-template DNA must thread through the gaps between the clamp subunits I & III and the clamp loader A and A’ domains. (4) Upon DNA binding in the interior chamber of the clamp loader, ATP hydrolysis is activated. (5) ATP hydrolysis at the B subunit breaks the interface at the AAA+ modules of the B and C subunits and allows the clamp to close around primer-template DNA. Further ATP hydrolyses at the C and D subunits dissolve the symmetric spiral of AAA+ modules, thus ejecting the clamp loader because the recognition of DNA and the clamp is broken. The clamp is now loaded onto primer-template DNA, and the clamp loader is free to recycle for another round of clamp loading.



Research conducted by: B.A. Kelch, D.L. Makino, and J. Kuriyan (University of California, Berkeley and the Howard Hughes Medical Institute) and M. O'Donnell (Rockefeller University and the Howard Hughes Medical Institute).

Research funding: U.S. 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: B.A. Kelch, D.L. Makino, M. O'Donnell, and J. Kuriyan, "How a DNA polymerase clamp loader opens a sliding clamp," Science 334, 1675 (2011).

ALS Science Highlight #249


ALSNews Vol. 331