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Updated: Jul 29, 2026
10:14
Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Published on: October 25, 2014
ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C
Y Shiomi1, J Usukura, Y Masamura
1Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101, Japan.
Summary
Replication Factor C (RFC) undergoes structural changes upon ATP binding, transitioning from a closed to an open form. This transformation is crucial for loading the Proliferating Cell Nuclear Antigen (PCNA) clamp onto DNA during replication.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Proliferating Cell Nuclear Antigen (PCNA) is a eukaryotic DNA sliding clamp essential for DNA replication, ensuring DNA polymerase engagement at the replication fork.
- Replication Factor C (RFC) is a heteropentameric complex responsible for loading PCNA onto primer DNA, a process requiring RFC's ATPase activity.
Purpose of the Study:
- To investigate the structural dynamics of RFC in response to ATP binding and hydrolysis.
- To elucidate the mechanism by which RFC facilitates the formation of the PCNA clamp on DNA.
Main Methods:
- Partial proteolysis sensitivity assays to detect structural changes in RFC upon nucleotide binding.
- Direct observation using electron microscopy to visualize RFC structures in different nucleotide-bound states.
Main Results:
- RFC exhibits distinct structural changes upon binding ATP, ATPgammaS, or ADP, with ATP inducing the most significant transformation.
- Electron microscopy revealed RFC exists in a closed 'U' form without ATP, converting to an open 'C' form upon ATP addition.
- PCNA is observed to be held within RFC, and the ATP-induced structural change in RFC is proposed to facilitate PCNA clamp formation on DNA.
Conclusions:
- RFC undergoes ATP-dependent conformational changes, transitioning to an open state that is critical for its function.
- The observed structural dynamics of RFC are hypothesized to be the driving force behind the loading and activation of the PCNA sliding clamp onto DNA.
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