Roswell Park Cancer Institute, Department of Cancer Genetics, Buffalo, New York, USA.
J Virol. 2013 Dec;87(23):13020-8. doi: 10.1128/JVI.01606-13. Epub 2013 Sep 25.
The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1's DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.
EBV 的 EBNA1 蛋白使携带 oriP 的质粒在增殖细胞中高效复制和分离。EBNA1 募集起始识别复合物（ORC）在 oriP 的一个元件 DS（二联体对称）上建立复制起点；在另一个元件 FR（重复家族）上，EBNA1 结合到一系列位点，从这些位点将质粒锚定到宿主染色体上以实现有丝分裂稳定性。我们报告的实验得出结论，为了使质粒从 oriP 高效复制，EBNA1 与宿主染色体的锚定在间期核内也是必需的。缺乏染色体结合能力的 EBNA1 的 DNA 结合域在瞬时转染的 HEK293 细胞中被发现支持弱的 DS 特异性复制，其活性为野生型 EBNA1 的 17%。复制效率低不是由于 DNA 结合域未能将质粒保留在核内，因为在这些瞬时转染的细胞的核部分中以相似的量和完全回收了质粒。其染色体锚定结构域被 22 个氨基酸核小体结合结构域取代的 EBNA1 衍生物在支持 oriP 功能方面完全有效。这意味着 EBNA1 的 DNA 结合域能够募集 ORC 到 DS，但如果质粒被锚定到宿主染色体上，该步骤或随后的复制才会有效。最后，在某些细胞系中，即使没有 FR，DS 也几乎无法支持瞬时质粒复制。从核中丢失缺乏 FR 的质粒并不能解释这一要求，这表明 FR 对染色体的更强锚定对于这些细胞的核内质粒复制是必需的。
