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The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes.

Original publication

DOI

10.1080/15384101.2015.1053667

Type

Journal article

Journal

Cell Cycle

Publication Date

03/08/2015

Volume

14

Pages

2509 - 2519

Keywords

Bimolecular Fluorescence Complementation, DNA Replication, Maf1, PCNA, Protein interactions, RNF7, SetD3, Binding Sites, Cell Line, Transformed, DNA Repair, DNA Replication, HEK293 Cells, High-Throughput Nucleotide Sequencing, Histone-Lysine N-Methyltransferase, Humans, Proliferating Cell Nuclear Antigen, Repressor Proteins, Ubiquitin-Protein Ligases