A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells.
A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells.
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DOI：
10.1080/15384101.2015.1053667
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发表时间：
2015-08-03
期刊：
影响因子：
--
通讯作者：
Green CM
中科院分区：
文献类型：
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作者：
Cooper SE;Hodimont E;Green CM
增殖细胞核抗原（PCNA）是DNA复制工厂的保守组分，并且与PCNA的相互作用介导许多必需的DNA复制酶到这些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是难以捉摸的，更好地了解它们将提高我们对如何实现基因组和表观遗传稳定性的理解。为了充分表征与PCNA相互作用的蛋白质组，我们开发了一种双分子荧光互补（BiFC）筛选人类细胞中的PCNA相互作用物。这种从人cDNA文库中筛选相互作用子的双杂交类型是快速和有效的。蛋白质相互作用的荧光读数使得能够容易地选择相互作用的克隆，并且我们将其与下一代测序相结合以鉴定编码相互作用蛋白质的cDNA。该方法能够重复鉴定先前表征的PCNA相互作用物，但重要的是还鉴定了RNF 7、Maf 1和SetD 3作为PCNA相互作用蛋白。我们验证了这些相互作用，从人类细胞提取物的免疫共沉淀和使用重组蛋白的相互作用分析。这些结果表明BiFC筛选是鉴定活哺乳动物细胞中蛋白质-蛋白质相互作用的有价值的方法。这种方法具有潜在的广泛应用，因为它是高通量和易于自动化。我们认为，考虑到这种与PCNA的相互作用，Maf 1，RNF 7和SetD 3可能参与DNA复制，DNA修复或相关过程。
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.