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ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load
X V Gomes1, S L Schmidt, P M Burgers
1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Abstract:
Binding of adenosine (3-thiotriphosphate) (ATPgammaS), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) was studied by filter binding. RFC alone bound 1.8 ATPgammaS molecules. However, when either PCNA or primer-template DNA were also present 2.6 or 2.7 ATPgammaS molecules, respectively, were bound. When both PCNA and DNA were present 3.6 ATPgammaS molecules were bound per RFC. Order of addition experiments using surface plasmon resonance indicate that RFC forms an ATP-mediated binary complex with PCNA prior to formation of a ternary DNA.PCNA.RFC complex. An ATP-mediated complex between RFC and DNA was not competent for binding PCNA, and the RFC.DNA complex dissociated with hydrolysis of ATP. Based on these experiments a model is proposed in which: (i) RFC binds two ATPs (RFC.ATP(2)); (ii) this complex binds PCNA (PCNA.RFC.ATP(2)), which goes through a conformational change to reveal a binding site for one additional ATP (PCNA.RFC.ATP(3)); (iii) this complex can bind DNA to yield DNA.PCNA.RFC.ATP(3); (iv) a conformational change in the latter complex reveals a fourth binding site for ATP; and (v) the DNA.PCNA.RFC.ATP(4) complex is finally competent for completion of PCNA loading and release of RFC upon hydrolysis of ATP.
Insights
Replication factor C (RFC) binds more ATPgammaS when combined with PCNA and DNA. This binding facilitates the formation of a ternary complex, crucial for PCNA loading and RFC release.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein-DNA Interactions
Background:
- Replication factor C (RFC) is essential for DNA replication.
- RFC interacts with PCNA and DNA to facilitate DNA polymerase processivity.
Purpose of the Study:
- To investigate the binding stoichiometry of ATPgammaS to RFC in the presence of PCNA and DNA.
- To elucidate the order of complex formation between RFC, PCNA, and DNA.
Main Methods:
- Filter binding assays to quantify ATPgammaS binding to RFC.
- Surface plasmon resonance (SPR) to study the kinetics of complex formation.
Main Results:
- RFC alone bound 1.8 ATPgammaS molecules.
- PCNA or DNA increased RFC binding to 2.6 or 2.7 ATPgammaS molecules, respectively.
- Both PCNA and DNA together increased RFC binding to 3.6 ATPgammaS molecules, indicating sequential binding events.
Conclusions:
- RFC forms an ATP-mediated binary complex with PCNA before binding DNA.
- A model is proposed where RFC sequentially binds up to four ATP molecules during the formation of the DNA.PCNA.RFC complex, which is competent for PCNA loading.