title: Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation.
abstract: The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.
authors.affiliation: Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel; and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Dovrat
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Stodola
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: Peter M J
authors.initials: PM
authors.lastname: Burgers
authors.affiliation: Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel; and aaharoni@bgu.ac.il.
authors.forename: Amir
authors.initials: A
authors.lastname: Aharoni
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D008565:Membrane Proteins; C022262:Okazaki fragments; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D000123:Acetyltransferases; C107118:ELO2 protein, S cerevisiae; D004258:DNA Polymerase III; D011088:DNA Ligases; D000072481:DNA Ligase ATP