Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 111; no. 39; pp. 14118 - 14123
• Main Authors: Dovrat, Daniel, Stodola, Joseph L., Burgers, Peter M. J., Aharoni, Amir
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.09.2014; National Acad Sciences
• Subjects: Acetyltransferases - metabolism; Amino Acid Substitution; Antigens; Binding Sites; Biochemistry; Biological Sciences; Cell nucleus; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA Ligase ATP; DNA Ligases - metabolism; DNA polymerase; DNA Polymerase III - metabolism; DNA Repair; DNA Replication; DNA, Fungal - chemistry; DNA, Fungal - genetics; DNA, Fungal - metabolism; Enzymes; Eukaryotes; Gels; Kinetics; Membrane Proteins - metabolism; Models, Biological; Monomers; Mutagenesis, Site-Directed; Plasmids; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - genetics; Proliferating Cell Nuclear Antigen - metabolism; Protein Binding; Protein Stability; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Trimers
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1321349111

Significance Proliferating cell nuclear antigen (PCNA) is a homotrimeric DNA sliding clamp that coordinates multiple DNA replication and repair processes by orchestrating the activity of various essential proteins. PCNA can bind up to three partners simultaneously, but despite extensive research, the functional significance of PCNA's trimeric structure remains unclear. We developed a novel approach for the generation of PCNA heterotrimers that contain both wild-type and mutant monomers. Using these heterotrimers, we show that PCNA can efficiently coordinate the activities of the three enzymes involved in Okazaki fragment maturation without binding them simultaneously. In contrast to the previously suggested “toolbelt” model for PCNA function, our results demonstrate sequential binding and release of partners on the PCNA trimer during complex biological processes. 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.