title: Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C.
abstract: DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.
authors.affiliation: 1600 SW Archer Road, P.O. Box 100245, Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL 32610-0245, USA.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Marzahn
authors.affiliation: 1600 SW Archer Road, P.O. Box 100245, Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL 32610-0245, USA.
authors.forename: Jaclyn N
authors.initials: JN
authors.lastname: Hayner
authors.affiliation: 1600 SW Archer Road, P.O. Box 100245, Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL 32610-0245, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Meyer
authors.affiliation: 1600 SW Archer Road, P.O. Box 100245, Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL 32610-0245, USA. Electronic address: lbloom@ufl.edu.
authors.forename: Linda B
authors.initials: LB
authors.lastname: Bloom
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051818:Replication Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D003374:Coumarins; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D000255:Adenosine Triphosphate; D004247:DNA; D051818:Replication Protein C
keywords: AAA+ATPase; Clamp loader; DNA replication; Proliferating cell nuclear antigen (PCNA); Replication factor C (RFC); Sliding clamp