The long-term goal of this proposal is to better define dynamic protein-DNA and protein-protein interactions in DNA replication. The system that will be examined in detail is the loading of the sliding clamp (processivity factor) onto DNA by the multi-subunit clamp loader complex. The investigator proposes to examine this system from 2 different organisms, E. coli and T4.
In Specific Aim 1, the investigator will examine how varying the structure of the DNA (DNA primer-template, RNA primer-template, blunt end, etc.) affects loading of the sliding clamp (beta) by the clamp loader complex (gamma complex). A novel fluorescence anisotropy-based binding assay will be used to identify protein-protein and protein-DNA interactions. A variety of different sub-complexes that can be isolated from pol III will be examined, including th tau complex. ATP dependent steps will be identified.
In Specific Aim 2, pre-steady-state kinetic approaches will be used to define rate-limiting processes in loading and the order of events in loading. Interactions between beta and gamma will be examined, along with protein-DNA interactions. Steps that require either ATP binding or hydrolysis will be identified. Pre-steady-state primer extension assays will be used to differentiate between productive and non-productive beta-DNA complexes.
In Specific Aim 3, similar experiments will be performed using the sliding clamp protein (gp45) and clamp loader complex (gp44/62) from phage T4 in order to define mechanistic similarities and differences between the two systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM055596-01A2
Application #
2697723
Study Section
Biochemistry Study Section (BIO)
Project Start
1998-07-01
Project End
2003-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
188435911
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Paschall, Christopher O; Thompson, Jennifer A; Marzahn, Melissa R et al. (2011) The Escherichia coli clamp loader can actively pry open the ?-sliding clamp. J Biol Chem 286:42704-14
Bloom, Linda B (2009) Loading clamps for DNA replication and repair. DNA Repair (Amst) 8:570-8
Anderson, Stephen G; Thompson, Jennifer A; Paschall, Christopher O et al. (2009) Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex. Biochemistry 48:8516-27
Thompson, Jennifer A; Paschall, Christopher O; O'Donnell, Mike et al. (2009) A slow ATP-induced conformational change limits the rate of DNA binding but not the rate of beta clamp binding by the escherichia coli gamma complex clamp loader. J Biol Chem 284:32147-57
Maher, Robyn L; Bloom, Linda B (2007) Pre-steady-state kinetic characterization of the AP endonuclease activity of human AP endonuclease 1. J Biol Chem 282:30577-85
Hariharan, Chithra; Bloom, Linda B; Helquist, Sandra A et al. (2006) Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies. Biochemistry 45:2836-44