Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.
Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.
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牛乳头瘤病毒 1 型 E1 蛋白氨基末端区域的分离，保留了起点结合和 E2 相互作用能力。
DOI：
10.1128/jvi.71.1.848-852.1997
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Wilson,VG
中科院分区：
文献类型：
--
作者：
Leng,X;Ludes-Meyers,JH;Wilson,VG
在体外DNA结合的结果从一系列的E1蛋白含有氨基末端或羧基末端截短表明，氨基酸121和284之间的序列是至关重要的起源结合。用含有内部、框内插入或缺失的E1蛋白进行的另外的结合实验证实了由截短的E1蛋白限定的区域的重要性，并且还证明了在全长E1蛋白的背景下不需要下游序列来进行结合活性。根据E1突变体的作图结果，构建了一个克隆（pE 1（121-311）），它在DNA结合的关键序列的近似边界内表达E1氨基酸。E1（121-311）蛋白保留了来源特异性DNA结合，证实了该区域不仅是必要的，而且对于来源识别也是足够的。E1（121-311）除与起始位点结合外，还与E2蛋白以冷敏感方式结合。因此，DNA结合和E2结合活性共定位于来自E1蛋白的氨基末端一半的191个氨基酸的功能结构域。最后，在该区域具有突变的三种E1蛋白都缺乏DNA结合活性，并且都在体内复制中有缺陷。这些E1突变体中的两个保留E2结合能力，表明E1的起点识别对于复制是至关重要的，并且不一定通过与E2蛋白的相互作用来拯救。
In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.