The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

Escherichia coli γ complex clamp loader functions to load the β sliding clamp onto sites of DNA replication and repair. The clamp loader uses the energy of ATP binding and hydrolysis to drive conformational changes allowing for β binding and opening, DNA binding, and then release of the β·DNA complex. Although much work has been done studying the sliding clamp and clamp loader mechanism, kinetic analysis of the events following β·γ complex·DNA formation is not complete. Using fluorescent clamp closing and release assays, we show that β closing is faster than β release, indicating that γ complex closes β before releasing it around DNA. Using a fluorescent ATP hydrolysis assay, we show that there is a burst of ATP hydrolysis before β closing and that β release may be the rate-limiting step in the overall clamp loading reaction. The combined use of these fluorescent assays provides a unique perspective into the E. coli clamp loader by providing a measure of the relative timing of different events in the clamp loading reaction, helping to elucidate the complicated clamp loading mechanism. Background: The γ complex loads the ring-shaped β sliding clamp onto DNA. Results: The rate of β clamp closing is faster than the rate of β release on DNA, and the first turnover of ATP hydrolysis is faster than β closing. Conclusion: Clamp closing occurs before clamp release but after a burst of ATP hydrolysis. Significance: These results demonstrate that clamp release around DNA is a two-step process.