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.
