Date of Award
DNA replication, clamp loaders, ATP hydrolysis, clamp proteins
DNA replicases utilize ring-shaped sliding clamps to ensure polymerase processivity. An ATP-dependent clamp loader topologically links the clamp around DNA in a multi-step mechanism. Clamp loaders are ring-shaped pentamers of AAA+ subunits. The pentamer complex contains 3-4 ATP sites, each located at the interface of two subunits. In each ATP site, an arginine residue from one subunit is located near the Î³-phosphate of ATP bound to the adjacent subunit. These arginines act as â€œarginine fingersâ€ that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. This thesis utilizes mutations in the arginine fingers of the E. coli and S. cerevisiae clamp loaders in order to examine steps in the clamp loading mechanism after the clamp loader binds ATP. The E. coli Î³ complex couples ATP hydrolysis to the loading of Î² sliding clamps onto DNA. We demonstrate that the Î´' subunit of Î³ complex contributes an arginine finger into ATP site D. Hydrolysis in site D is inhibited by mutation of the Î³ subunit arginine finger, which affects sites B and C. The Î´' arginine finger also mediates Î² binding and the Î³ arginine fingers are important for DNA binding. Utilizing a three-subunit fusion construct, we determined that a single arginine finger mutation in either site B or C causes inactivation of Î³ complex. Replication factor C (RFC) loads the processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. ATP binding to RFC activates a Î³-phosphate sensor in ATP site C that promotes DNA association by RFC-PCNA. DNA binding to the RFC-PCNA complex then triggers the ATP hydrolysis cycle which starts in site C and ends with ATP site D. ATP hydrolysis in site D is uniquely stimulated by PCNA and we propose that this site is coupled to closure of PCNA around DNA. PCNA closure severs contact to RFC subunits D and E, and the Î³- phosphate sensor of ATP site C is switched off, leading to ejection of RFC from the site of PCNA loading. This work sheds light on conserved aspects of ATP site function that may extend to all clamp loaders.
Johnson, Aaron M., "Comparative Studies of Processivity Clamp Loader ATP Site Function." (2007). Student Theses and Dissertations. 15.