Specificity and efficiency of editing of mismatches involved in the formation of base-substitution mutations by the 3'----5' exonuclease activity of phage T4 DNA polymerase.
Specificity and efficiency of editing of mismatches involved in the formation of base-substitution mutations by the 3'----5' exonuclease activity of phage T4 DNA polymerase.
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通过噬菌体 T4 DNA 聚合酶的 3----5 核酸外切酶活性，编辑参与碱基取代突变形成的错配的特异性和效率。
DOI：
10.1073/pnas.84.4.915
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发表时间：
1987
影响因子：
11.1
通讯作者：
Sinha,NK
中科院分区：
文献类型：
--
作者：
Sinha,NK
用一种敏感的感染性测定法测定了噬菌体T4 DNA聚合酶的3 '----5'核酸外切酶活性对碱基错配编辑的特异性和效率。化学合成了一系列寡脱氧核苷酸引物链。当这些引物与含有琥珀密码子的phi X174单链DNA杂交时，在琥珀密码子内引物链的3 ′-羟基末端产生错配核苷酸。在这些引物X模板上的DNA合成而不去除错配的末端核苷酸导致异源双链体分子的形成，所述异源双链体分子在转染到琥珀非抑制宿主中时产生活的回复突变体。该方法允许将错配的编辑效率确定为逃避编辑的1/10（6）错配，并允许研究可以在琥珀密码子处产生可行回复突变体的所有八个错配。引物与phi X 174、am 16和am3密码子杂交的实验结果表明，T4 DNA聚合酶错配编辑的顺序为T模板× G引物小于（A × G，T × C）小于（T × T，G × A，G × G，A × C）小于A × A。编辑的效率取决于错配以及相邻的DNA序列。在这些合成条件下，3 ′-5 ′核酸外切酶的活性，取决于错配和最近邻以外的DNA序列，估计对T4 DNA聚合酶的准确性的贡献为2.3 × 10（3）至大于10（6）倍。
The specificity and efficiency of base-mispair editing by the 3'----5' exonuclease activity of phage T4 DNA polymerase has been measured using a sensitive infectivity assay. A series of oligodeoxynucleotide primer chains was synthesized chemically. These primers, when hybridized to phi X174 single-stranded DNAs containing an amber codon, result in a mispaired nucleotide at the 3'-hydroxyl end of the primer chain within the amber codon. DNA synthesis on these primer X templates without the removal of the mispaired terminal nucleotide results in the formation of heteroduplex molecules that yield viable revertants upon transfection into an amber nonsuppressor host. This method permits determination of the efficiency of editing of a mismatch to 1 in 10(6) mismatches that escape editing and allows all eight mispairs that can yield viable revertants at an amber codon to be studied. The results of experiments with primers hybridizing to phi X174 am16 and am3 codons show that the order of mispair editing by T4 DNA polymerase is Ttemplate X Gprimer less than (A X G, T X C) less than (T X T, G X A, G X G, A X C) less than A X A. The efficiency of editing depends upon the mispair, as well as the neighboring DNA sequence. Under these conditions of synthesis, the 3'----5' exonuclease activity, depending upon the mispair and DNA sequences beyond the nearest neighbors, is estimated to contribute a factor of from 2.3 X 10(3)- to greater than 10(6)-fold to the accuracy of T4 DNA polymerase.