PROJECT SUMMARY The maintenance of genome stability across generations is critical for human health and relies on the efficient repair of spontaneous DNA lesions and the faithful duplication of all chromosomal DNA prior to cell division. The highly conserved DNA sliding clamps PCNA and 9-1-1 are critical for these genome maintenance mechanisms in eukaryotic cells by acting as mobile hubs for the assembly of the protein complexes mediating the signaling and repair of DNA damage and conducting the faithful replication of the nuclear chromosomes. The dynamic association of PCNA and 9-1-1 with chromosomal DNA is controlled by a set of four conserved and related ATP-dependent clamp loader complexes that each perform non-redundant genome maintenance functions in the cell. How eukaryotic clamp loaders target their client clamps to sites of DNA replication and repair and load the clamps around DNA has been the subject of intense investigation for several decades. Using advanced biochemical reconstitution approaches and cryogenic electron-microscopy (cryo-EM), we have recently determined the first structures of active eukaryotic clamp loader:clamp:DNA complexes, which revealed the molecular basis for the substrate specificities of the yeast RFC and Rad24-RFC complexes and resulted in a significant revision of current clamp loading models. Building on this work, here we propose to extend and advance the approaches established by us for the yeast RFC:PCNA and Rad24-RFC:9-1-1 clamp loader systems to characterize the molecular mechanisms of the yeast and human orthologues of Ctf18- RFC:PCNA, Elg1ATAD5-RFC:PCNA, and RAD17-RFC:9-1-1. The innovative approach leverages the expertise of Dr. Remus’ laboratory in the biochemical reconstitution of eukaryotic DNA replication and the expertise of Dr. Hite’s laboratory in the characterization of the conformational landscapes of protein complexes by cryo-EM. The proposed studies will determine the mechanistic basis for the functional specialization of CTF18-RFC (Aim 1), uncover the currently unknown mechanism of PCNA unloading by Elg1ATAD5-RFC:PCNA (Aim 2) and visualize the spectrum of conformational states of the human RAD17-RFC:9-1-1 complex to reveal the evolutionary conservation or divergence of the 9-1-1 checkpoint clamp loading mechanism (Aim 3). Collectively, this work will provide novel mechanistic insight into fundamental genome maintenance pathways.