| Sliding clamps are ring-shaped protein complexes that encircle DNA and tether to DNA polymerase, keeping the polymerase in contact with the DNA and thus enabling rapid and processive DNA replication. Sliding clamps are conserved throughout all domains of life, and are placed around DNA in an ATP-dependent reaction by a machine-like protein complex known as the clamp loader. The eukaryotic clamp for DNA replication is the homotrimeric PCNA. Another type of clamp, the Rad9-Hus1-Rad1 (9-1-1) complex, is a heterotrimer that shares homology to PCNA. The 9-1-1 complex is a damage sensor in the DNA damage checkpoints, which are signaling processes that stop progression of the cell cycle in response to DNA damage.;This dissertation addresses the function and loading of sliding clamps in four chapters. Chapter 1 provides an introduction to the structure and function of sliding clamps in DNA replication, DNA repair, and the DNA damage checkpoints, as well as to the structural features of clamp loaders and the mechanism of the clamp loading process. Chapter 2 examines the archaeal clamp loader, which resembles those of eukaryotes. Truncation of a C-terminal extension of the large subunit of the clamp loader shows that the extension is not necessary for clamp loader function, and mutational analysis of the clamp loader shows that the DNA-binding motif used by eukaryotic and prokaryotic clamp loaders is conserved in archaea. In Chapter 3, the 25 A three-dimensional negative-stain electron microscopy reconstruction of the yeast homologue of 9-1-1 is presented, which reveals that the 9-1-1 homologue has a similar ring-shaped structure to PCNA but is larger in all dimensions. Finally, Chapter 4 presents two x-ray crystal structures of primer-template DNA bound to the center of PCNA. A model of DNA extending through PCNA during clamp loading is constructed, which suggests that PCNA-DNA contacts are made during clamp loading that are similar to those observed in one of the PCNA-DNA structures. A number of residues in this proposed DNA binding site in the center of PCNA are mutated and are shown to contribute to the clamp loading process. |
