Student Theses and Dissertations

Date of Award

2007

Document Type

Thesis

RU Laboratory

O'Donnell Laboratory

Keywords

DNA replication, clamp loaders, ATP hydrolysis, clamp proteins

Abstract

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 γ-phosphate of ATP bound to the adjacent subunit. These arginines act as “arginine fingers” 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 γ complex couples ATP hydrolysis to the loading of β sliding clamps onto DNA. We demonstrate that the δ' subunit of γ complex contributes an arginine finger into ATP site D. Hydrolysis in site D is inhibited by mutation of the γ subunit arginine finger, which affects sites B and C. The δ' arginine finger also mediates β binding and the γ 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 γ complex. Replication factor C (RFC) loads the processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. ATP binding to RFC activates a γ-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 γ- 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.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/126

Download

Included in

Life Sciences Commons

Share

COinS