Birkenbihl R P, Kemper B
Institut für Genetik der Universität zu Köln, Zülpicherstrasse 47, Köln, D-50674, Germany.
J Mol Biol. 1998 Jul 3;280(1):73-83. doi: 10.1006/jmbi.1998.1851.
Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4. Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA. In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated. Some of these mutations affected the dimerization of Endo VII. Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state. Only the dimeric fraction of this protein bound to DNA. The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde. The dimerization domain was thus determined to reside between amino acid residues 55 and 105. Computer analyses predicted two alpha-helices (H2 and H3) in this section of the protein. As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization. Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them.
核酸内切酶VII（Endo VII）是噬菌体T4的一种Holliday结构解离酶。其核酸水解活性取决于子活性，按执行顺序依次为：（i）二聚化，（ii）与DNA结合，（iii）DNA切割。为了将这些子功能分配到该蛋白质的一级序列中，分离出了一系列DNA切割缺陷的自发点突变。其中一些突变影响了Endo VII的二聚化。与在溶液中完全二聚化的野生型蛋白质相比，其中一种突变蛋白（W87R）超过95%保持单体状态。只有该蛋白质的二聚体部分与DNA结合。通过从两端截短基因并通过与戊二醛交联分析纯化肽的二聚化能力，对Endo VII的二聚化结构域进行了定位。因此确定二聚化结构域位于氨基酸残基55和105之间。计算机分析预测该蛋白质的这一部分有两个α螺旋（H2和H3）。如异源二聚体形成所示，二聚化需要两个H3螺旋拷贝，但只需要一个H2螺旋拷贝。螺旋轮分析表明，两个螺旋沿其轴都暴露一个疏水表面，这表明H3螺旋之间的疏水相互作用介导了Endo VII二聚体的形成，而H2螺旋使其稳定。
