Truniger Verónica, Lázaro José M, Salas Margarita
Instituto de Biología Molecular Eladio Viñuela, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Nucleic Acids Res. 2004 Jan 16;32(1):361-70. doi: 10.1093/nar/gkh184. Print 2004.
The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of phi29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be involved in DNA and TP binding. Therefore, we have studied the role of the C-terminus of phi29 DNA polymerase by deletion of the last 13 residues of this enzyme. This fragment includes a previously defined region conserved in family B DNA polymerases. The resulting DNA polymerase Delta13 was strongly affected in DNA binding, resulting in a distributive replication activity. Additionally, the capacity of the truncated polymerase to interact with TP was strongly reduced and its initiation activity was very low. On the other hand, its nucleotide binding affinity and its fidelity were not affected. We propose that the C-terminal 13 amino acids of phi29 DNA polymerase are involved in DNA binding and in a stable interaction with the initiator protein TP, playing an important role in the intrinsic processivity of this enzyme during polymerization.
拇指亚结构域位于多种B族DNA聚合酶的C末端区域，其晶体结构显示，在结合DNA时会发生移动，改变构象以几乎完全包裹DNA。因此，它参与了DNA结合。与此一致的是，对phi29 DNA聚合酶的部分蛋白酶解研究表明，在存在DNA或末端蛋白（TP）的情况下，位于其C末端区域的切割位点的可及性降低，这表明在底物结合时该区域会发生构象变化，并暗示该区域可能参与DNA和TP的结合。因此，我们通过缺失该酶的最后13个残基来研究phi29 DNA聚合酶C末端的作用。该片段包括一个先前定义的在B族DNA聚合酶中保守的区域。产生的DNA聚合酶Delta13在DNA结合方面受到强烈影响，导致分布性复制活性。此外，截短的聚合酶与TP相互作用的能力大大降低，其起始活性非常低。另一方面，其核苷酸结合亲和力和保真度不受影响。我们提出，phi29 DNA聚合酶的C末端13个氨基酸参与DNA结合以及与起始蛋白TP的稳定相互作用，在该酶聚合过程中的内在持续性中发挥重要作用。
