Reverse-chaperoning activity of an AAA+ protein
Cheng Liu1, Mary C McKinney, Yi-Hsing Chen
1Department of Physics and the Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Champaign, Illinois, USA.
Biophysical Journal
|March 1, 2011
Summary
The archaeal clamp loader (RFC) unexpectedly assembles the sliding clamp (PCNA) from monomers. This RFC-mediated PCNA assembly is ATP-dependent and results in a functional clamp for DNA replication.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Replicative DNA polymerases achieve high speed and processivity by interacting with sliding clamps.
- Sliding clamps, like PCNA, form closed rings that must be opened by clamp loader proteins (e.g., RFC) before loading onto DNA.
- Clamp loaders are AAA+ ATPases crucial for DNA replication initiation.
Purpose of the Study:
- To investigate the mechanism of clamp loading by RFC and the function of PCNA from the archaeon Methanosarcina acetivorans.
- To characterize the interaction between RFC and PCNA using real-time fluorescence assays.
- To explore novel functions of AAA+ proteins in protein complex assembly.
Main Methods:
- Development of real-time fluorescence assays to monitor PCNA and RFC interactions.
- Utilizing motion-based DNA polymerization assays to assess clamp functionality.
- Biochemical characterization of RFC's ATP-bound state and its role in PCNA assembly.
Main Results:
- RFC from Methanosarcina acetivorans demonstrated an unexpected ability to assemble PCNA rings from individual monomers in solution.
- The RFC-assembled PCNA rings were confirmed to be functional in DNA polymerization.
- This PCNA assembly activity was dependent on the ATP-bound conformation of RFC.
Conclusions:
- RFC exhibits a novel reverse-chaperoning activity, templating the assembly of the PCNA ring.
- AAA+ proteins can play diverse roles beyond canonical clamp loading, including facilitating protein complex formation.
- This finding provides new insights into the regulation of DNA replication machinery and protein assembly mechanisms.
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