Rodríguez Irene, Lázaro José M, Salas Margarita, De Vega Miguel
Instituto de Biología Molecular "Eladio Viñuela", Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
J Mol Biol. 2004 Apr 2;337(4):829-41. doi: 10.1016/j.jmb.2004.02.018.
By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx(2)h motif. Here, we have studied, by site-directed mutagenesis, the functional role of phi29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that phi29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.
通过对蛋白质引发的DNA聚合酶的真核生物类型（B家族）亚组中的DNA聚合酶进行多序列比对，我们鉴定出五个带正电荷的氨基酸，它们在（S/T）Lx(2)h基序的N端特别保守。在这里，我们通过定点诱变研究了phi29 DNA聚合酶残基Arg96、Lys110、Lys112、Arg113和Lys114在依赖蛋白质引发事件的特定反应中的功能作用。在残基Arg96、Arg113和Lys114处以及在较低程度上在Lys110和Lys112处引入的突变显示出蛋白质引发起始步骤存在缺陷。对突变衍生物R96A、K110A、K112A、R113A和K114A与双链DNA和末端蛋白（TP）的相互作用进行分析后，我们得出结论：phi29 DNA聚合酶残基Arg96是一个重要的DNA/TP配体残基，对于形成稳定的DNA聚合酶/DNA(TP)复合物至关重要，而残基Lys110、Lys112和Arg113可能在DNA复制第一步中与TP-DNA模板建立接触时发挥作用。我们还讨论了残基Lys114对于形成功能活性DNA聚合酶/TP复合物的重要性。这些结果，连同那些残基在蛋白质引发的DNA聚合酶中的高度保守性，强烈表明这些氨基酸在与DNA聚合酶底物DNA和TP建立适当相互作用以成功完成TP-DNA复制的第一步中具有功能作用。
