Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA.
Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA.
复制标题
人类免疫缺陷病毒 I 型逆转录酶复制天然 DNA 的保真度。
DOI：
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发表时间：
1989
影响因子：
14.9
通讯作者：
Frank Grosse
中科院分区：
文献类型：
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作者：
J. Weber;Frank Grosse
在大肠杆菌中表达人免疫缺陷病毒Ⅰ型（HIV-1）逆转录酶。coli中，并纯化至接近均一。该酶含有逆转录酶、DNA聚合酶和核糖核酸酶H活性。DNA聚合酶活性将单引物phi X174（+）DNA转化为双链形式。三分之二的复制产物可连接到共价闭合的环状DNA（RFIV形式DNA），表明通过HIV逆转录酶的DNA合成可以进行，直到酶匹配预先存在的引物分子的5 '末端。用phi X174 am 16逆转录试验测定HIV逆转录酶的体外准确度为1/7，400。根据合并偏倚研究确定单个错配的回复率为：dGMP：T模板错配为1/8，000，dGMP：A模板错配为1/35，000，dAMP：G模板错配为1/45，000，dCMP：T模板错配为1/73，000，dCMP：A模板错配为1/140，000，dGMP：G模板错配为1/180，000。dTMP：T模板错配低于测定的检测限，表明该特定错配的回复率小于1/300，000。
Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.