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'irrrnTTiiiiiniiiiiiiiiriim proteins, the DNA polymerases that act at the replication fork are only able to synihesize 20-100 base pairs before releasing from the template. How is the processivity of these enzymes increased so dramatically at the replication fork?

The key to the high processivity of the DNA polymerases that act at replication forks is their association with proteins called sliding DNA clamps. These proteins are composod of multiple identical subunits that assemble in the shape of a "doughnut." The hole in the center of the clamp is large enough to encircle the DNA double helix and leave room for a layer of one or two water molecules between I he DNA and the protein (Figure 8-173). These clump proteins slide along the DNA without dissociating from it. Sliding DNA clamps also bind tightly to DNA polymerases at replication forks. Thus, the clamp encircles the newly synthesized double-stranded DNA and the polymerase associates with the primer:template junction (Figure 8-17b). This complex between the polymerase and the sliding clamp moves efficiently along the DNA lemplate during DNA synthesis.

How does the association with the sliding clamp change the processivity of the DNA polymerase? In the absence of the sliding clamp, a DNA polymerase dissociates and diffuses away from the

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FICUflE 8-17 Structure of a sliding DNA clamp, (a) Three-dimensional structure of a sliding DNA damp associated with DMA. The opening through the center of the sliding damp is about 35 angstroms and the width of the DNA helix is approximately 20 angstroms. This provides enough space to allow a thin layer of one or two water motecules between the sliding damp and the DNA. This is thought to allow the damp to slide along the DNA easily (Knshna IS.. Kong X.P., Gary S„ Burgers, PM, and Kuriyan J 1994. Cell 79: 1233 ) Image prepared with BobScnpt, MolSaipt and Raster 3D (b) Sliding DNA clamps encircle the newly replicated DN'A produced by an associated DNA polymerase. The sliding clamp interacts with the part of the DNA polymerase that is closest to the newly synthesized DNA as it emerges from the DNA polymerase.

template DNA on average once every 20-100 base pairs synthesized. In the presence of the sliding clamp, the DNA polymerase still disengages its active site from the 3'OM end of the DNA frequently, but the association with the sliding clamp prevents the polymerase from diffusing away from the DNA (Figure 8-18). By keeping the DNA polymerase in close proximity to the DNA, the sliding clamp ensures that the DNA polymerase rapidly rebinds the same primentemplate junction, vastly increasing the processivity of the DNA polymerase.

Once an ssDNA template is completely copied, the DNA polymerase must be released from this DNA and the sliding clamp to act at a new primentemplate junction. This release is accomplished by a change in the affinity between the DNA polymerase and the sliding clamp that depends on the bound DNA. DNA polymerase bound to a primer:template junction has a high affinity for the clamp. In contrast, when the DNA polymerase reaches the end of an ssDNA template (for example, at the end of an Okazaki fragment), a change in the conformation of the DNA polymerase reduces its affinity for the sliding clamp and the DNA (see Figure 8-18). Thus, when a polymerase completes the replication of a stretch of DNA, it is released by the sliding clamp so it can act at a new prjmertemplate junction. The clamp, on the. other hand, remains bound to the DNA and can bind other enzymes that act on the newly synthesized DNA (as we describe below).

FIGURE 8-18 Sliding DNA damps increase the processivity of associated DNA polymerases.

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Once released from a DNA polymerase, sliding clamps are not immediately removed from the replicated DNA. Instead, other proteins thai must function at the site of recent DNA synthesis to perform their function interact with the clamp proteins. As described in Chapter 7, enzymes that assemble chromatin in eukaryotic cells are recruited to the sites of DNA replication by an interaction with the eukaryotic sliding DNA clamp (called PCNA). Similarly, eukaryotic proteins involved in Okazaki fragment repair also interact with sliding clamp proteins. In each case, by interacting with sliding clamps, these proteins accumulate at sites of new DNA synthesis where they are needed the most.

Sliding clamp proteins are a conserved part of the DNA replication apparatus derived from organisms as diverse as viruses, bacteria, yeast, and humans. Consistent with their conserved function, the structure of sliding clamps derived from these different organisms is also conserved (Figure 6-19). In each case, the clamp bas the same sixfold symmetry and the same diameter. Despite the similarity in overall structure, however, the number of subunits that come together to form the clamp differs.

Sliding Clamps Are Opened and Placed on DNA by Clamp Loaders

The sliding clamp is a closed ring in solution and must open to encircle the DNA double helix. A special class of protein complexes, called sliding clamp loaders, catalyzes the opening and placement of sliding clamps on the DNA. These enzymes couple ATP binding and hydrolysis

FIGURE 8-19 The three-dimensional structure of sliding DNA damps isolated from different organisms. Slicing DNA clamps are found across all organisms and share a simitar structure (a) The sliding DNA damp from £ coll is composed of two copies of the p protein {Kong X.R. Onrust R.. CfDonnell M, and Kuriyan J. 1992. Cell 69: 425.) (b) TheT4 phage sliding DNA clamp is a tnmer of the gp45 protein, (fvtoarefi I, Jeruzalrm D, turner J, O'Donnell M_, and Kunyan J. 2002 JMol Biol 296. 1215.) (c) The eukaryotic sliding DNA clamp is a tnmer of the PC MA protein. (Krrshna T.S., Kong X.P., Gary 5.. Burgers P.M., and Kunyan J. 1994 Cell 79: 1233.) Images prepared with BobScnpt, MolScript, and Raster 3D.

FIGURE 8-19 The three-dimensional structure of sliding DNA damps isolated from different organisms. Slicing DNA clamps are found across all organisms and share a simitar structure (a) The sliding DNA damp from £ coll is composed of two copies of the p protein {Kong X.R. Onrust R.. CfDonnell M, and Kuriyan J. 1992. Cell 69: 425.) (b) TheT4 phage sliding DNA clamp is a tnmer of the gp45 protein, (fvtoarefi I, Jeruzalrm D, turner J, O'Donnell M_, and Kunyan J. 2002 JMol Biol 296. 1215.) (c) The eukaryotic sliding DNA clamp is a tnmer of the PC MA protein. (Krrshna T.S., Kong X.P., Gary 5.. Burgers P.M., and Kunyan J. 1994 Cell 79: 1233.) Images prepared with BobScnpt, MolScript, and Raster 3D.

to the placement of ihe sliding clamp around primer.template junctions on the DNA [see Box 8-2, ATP Control of Protein Function). The clamp loader also removes sliding clamps from the DNA when they are no longer in use. Like DNA helicases and topoisomerases, these enzymes alter the conformation of their target (the sliding clamp) but not its chemical composition.

What controls when sliding clamps are loaded and removed from the DNA? Loading of a sliding clamp occurs anytime a primentem-plate junction is present in the cell. These DNA structures are formed not only during DNA replication but also during several DNA repair events (see Chapter 9). A sliding clamp can only be removed from the DNA if it is not being used by another enzyme. Sliding clamp loaders and DNA polymerases cannot interact with a sliding clamp at the same time because they have overlapping binding sites on the same face of the sliding clamp. Thus, a sliding clamp that is bound to a DNA polymerase is not subject to removal from the DNA. Similarly, nucleosome assembly factors, Okazaki fragment repair proteins, and other DNA repair proteins all interact with the same region of the sliding clamp as the clamp loader. Thus, sliding clamps are only removed from the DNA once all the enzymes that interact with them have completed their function.

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