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首页> 外文期刊>Cell cycle >A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells
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A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

机译：荧光双分子互补筛选揭示了MAF1，RNF7和SETD3作为人类细胞中PCNA相关蛋白

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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.
机译：增殖细胞核抗原（PCNA）是DNA复制工厂的保守组成部分，与PCNA的相互作用介导了许多必需的DNA复制酶向这些DNA合成位点的募集。对这些工厂的结构和组成的完整描述仍然难以捉摸，并且对它们的更好了解将增进我们对如何实现基因组维持和表观遗传稳定性的理解。为了全面表征与PCNA相互作用的蛋白质，我们开发了一种双分子荧光互补（BiFC）筛选技术，用于人细胞中PCNA相互作用物。这种来自人类cDNA文库中相互作用子的2杂交型筛选是快速有效的。蛋白质相互作用的荧光读数可以方便地选择相互作用的克隆，我们将其与下一代测序相结合，以鉴定编码相互作用蛋白的cDNA。该方法能够可再现地鉴定先前表征的PCNA相互作用物，但重要的是还鉴定了RNF7，Maf1和SetD3作为PCNA相互作用蛋白。我们通过从人细胞提取物中进行共免疫沉淀以及使用重组蛋白进行的相互作用分析，验证了这些相互作用。这些结果表明，BiFC筛选是鉴定活的哺乳动物细胞中蛋白质相互作用的一种有价值的方法。该方法具有高吞吐量并且易于自动化，因此具有潜在的广泛应用。我们建议，鉴于与PCNA的这种相互作用，Maf1，RNF7和SetD3可能参与DNA复制，DNA修复或相关过程。

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