Characterization of the interaction between the Baculovirus replication factors LEF-1 and LEF-2
Characterization of the interaction between the Baculovirus replication factors LEF-1 and LEF-2
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DOI：
10.1128/jvi.71.4.3114-3119.1997
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发表时间：
1997-04-01
影响因子：
5.4
通讯作者：
Rohrmann, GF
中科院分区：
文献类型：
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作者：
Evans, JT;Leisy, DJ;Rohrmann, GF
苜蓿夜蛾多核衣壳核型多角体病毒具有瞬时DNA复制所需的6个基因和3个刺激基因。我们使用酿酒酵母和谷胱甘肽S转移酶融合亲和力实验证明了其中两个基因的产物Lef-1和Lef-2在两个双杂交实验中相互作用。利用酵母双杂交实验，我们将Lef-2的相互作用结构域定位到第20位到60位之间的氨基酸。对Lef-1的广泛缺失分析未能揭示出一个界定的相互作用结构域，这表明可能存在必要的二级结构元件被这些缺失失活。所有不能相互作用的表达Lef-1和Lef-2的克隆也不能支持显著水平的瞬时DNA复制，这表明这种相互作用是DNA复制所必需的。对Lef-1基因进行序列分析，发现LEF-1基因有一个类似于启动酶的基序WVVDAD。当该基序突变为WVVQAD时，Lef-1不再支持瞬时DNA复制。
The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. when this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.