Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein
Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein
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DOI：
10.1073/pnas.0407818102
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发表时间：
2005-02-22
影响因子：
11.1
通讯作者：
McBride, AA
中科院分区：
文献类型：
--
作者：
Brannon, AR;Maresca, JA;McBride, AA
乳头瘤病毒E2蛋白在病毒转录调节、DNA复制和附加型基因组维持中起作用。病毒基因组通过E2蛋白附着于有丝分裂染色体而维持在分裂细胞中。为了研究E2的染色体束缚功能，在酿酒酵母中开发了质粒稳定性测定，以确定E2蛋白是否可以维持含有酵母自主复制序列复制元件但着丝粒元件被E2结合位点取代的质粒。E2表达不足以维持这样的质粒，但质粒稳定性可以通过表达哺乳动物蛋白Brd 4来挽救。在Brd 4和E2蛋白的存在下，具有多个E2结合位点的质粒是稳定的，无需选择。S.酿酒酵母编码一种名为Bdf 1的Brd 4同系物，它不含与E2蛋白相互作用的C-末端结构域。Bdf 1和Brd 4 C-末端“尾”的融合蛋白可以支持E2介导的质粒在酵母中的维持。使用一组突变的E2蛋白，我们确定质粒稳定性需要E2结合DNA和与Brd 4和哺乳动物有丝分裂染色体相互作用的能力，但不需要其复制起始和反式激活功能。色葡萄本文所述的基于病毒的质粒维持测定是用于剖析附加型病毒基因组复制机制和筛选参与质粒维持的其它宿主蛋白因子的宝贵工具。
The papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element but with the centromeric element replaced by E2-binding sites. E2 expression was not sufficient to maintain such plasmids, but plasmid stability could be rescued by expression of the mammalian protein Brd4. In the presence of both Brd4 and E2 proteins, plasmids with multiple E2-binding sites were stable without selection. S. cerevisiae encodes a homolog of Brd4 named Bdf1 that does not contain the C-terminal domain that interacts with the E2 protein. A fusion protein of Bdf1 and the Brd4 C-terminal "tail" could support E2-mediated plasmid maintenance in yeast. Using a panel of mutated E2 proteins, we determined that plasmid stability required the ability of E2 to bind DNA and to interact with Brd4 and mammalian mitotic chromosomes but did not require its replication initiation and transactivation functions. The S. cerevisiae-based plasmid maintenance assays described here are invaluable tools for dissecting mechanisms of episomal viral genome replication and screening for additional host protein factors involved in plasmid maintenance.