φ29 DNA polymerase-terminal protein interaction.: Involvement of residues specifically conserved among protein-primed DNA polymerases
φ29 DNA polymerase-terminal protein interaction.: Involvement of residues specifically conserved among protein-primed DNA polymerases
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DOI：
10.1016/j.jmb.2004.02.018
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发表时间：
2004-04-02
影响因子：
5.6
通讯作者：
de Vega, M
中科院分区：
文献类型：
--
作者：
Rodríguez, I;Lázaro, JM;de Vega, M
通过对来自真核生物型（B家族）蛋白质引发的DNA聚合酶亚组的DNA聚合酶的多重序列比对，我们已经鉴定了5个带正电荷的氨基酸，它们特别保守，位于（S/T）Lx（2）h基序的N-末端。在这里，我们已经研究了，通过定点诱变，功能作用的phi 29 DNA聚合酶残基Arg 96，Lys 110，Lys 112，Arg 113和Lys 114在特定的反应依赖于蛋白质引发事件。在残基Arg 96、Arg 113和Lys 114处引入的突变以及在较低程度上的Lys 110和Lys 112处引入的突变显示出有缺陷的蛋白质引发起始步骤。通过分析突变体衍生物R96 A、K110 A、K112 A、R113 A和K114 A与双链DNA和末端蛋白（TP）的相互作用，我们可以得出结论，phi 29 DNA聚合酶残基Arg 96是重要的DNA/TP-配体残基，是形成稳定的DNA聚合酶/DNA（TP）复合物所必需的，而残基Lys 110，Lys 112和Arg 113可能在DNA复制的第一步中与TP-DNA模板建立接触中起作用。残基Lys 114的重要性，使功能活性的DNA聚合酶/TP复合物进行了讨论。这些结果，连同这些残基之间的高度保守的蛋白质引发的DNA聚合酶，强烈建议的功能作用，这些氨基酸在建立适当的相互作用与DNA聚合酶底物，DNA和TP，成功地完成TP-DNA复制的第一步。(C)2004 Elsevier Ltd.保留所有权利。
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. (C) 2004 Elsevier Ltd. All rights reserved.