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首页> 外文期刊>Biochemistry >Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase
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Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase

机译：噬菌体T4抗突变体​​DNA聚合酶的动力学表征

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Fidelity of DNA replication by bacteriophage T4 DNA polymerase is achieved in a multiplicative process: base selection by its polymerase activity and removal of misincorporated nucleotides by its exonuclease activity. The wild-type polymerase is capable of maintaining a balance between the two activities so that DNA replication fidelity is maximized without excessive waste of nucleotidos. Antimutator enzymes exhibit a higher DNA replication fidelity than the wild-type enzyme, at the cost of increased nucleotide turnover. The antimutator A737V polymerase has been characterized kinetically using pre-steady-state and steady-state methods to provide a kinetic sequence which defines the effect of the mutation on the discrete steps controlling DNA replication fidelity, Comparison of this sequence to that of the wild type [Capson, L. T., Peliska, J, A., Kaboord, B. F., Frey, M. W., Lively, C., Dahiberg, M., and Benkovic, S, J. (1992) Biochemistry 31, 10984-10994] revealed that A737V polymerase differs in two ways. The rates at which DNA is transferred between the exonuclease and polymerase sites are reduced approximately 7-fold for a duplex DNA containing a mismatched 3'-terminus, and the partitioning of the mismatched duplex between the polymerase and exonuclease sites is 1:2 versus 4:1 for the wildtype enzyme. The exonuclease activity of A737V relative to the wild-type enzyme is unchanged on single-stranded DNA. However, the difference in partitioning the duplex DNA between the exonuclease and polymerase active sites results in an enhanced exonuclease activity for the antimutator enzyme. [References: 36]
机译：通过噬菌体T4 DNA聚合酶进行DNA复制的保真度是通过乘法过程实现的：通过其聚合酶活性进行碱基选择，并通过其核酸外切酶活性去除错误掺入的核苷酸。野生型聚合酶能够在两种活性之间保持平衡，从而使DNA复制的保真度最大化，而不会浪费过多的核苷酸。抗突变酶比野生型酶具有更高的DNA复制保真度，但核苷酸更新率却有所增加。抗突变体A737V聚合酶已使用稳态前和稳态方法进行了动力学表征，以提供动力学序列，该序列定义了突变对控制DNA复制保真度的离散步骤的影响，该序列与野生型的比较[Capson，LT，Peliska，J.，Kaboord，BF，Frey，MW，Lively，C.，Dahiberg，M。和Benkovic，S，J。（1992）Biochemistry 31，10984-10994]揭示了A737V。聚合酶在两个方面有所不同。对于含有错配的3'末端的双链体DNA，DNA在核酸外切酶和聚合酶位点之间转移的速率降低了约7倍，聚合酶和核酸外切酶位点之间错配的双链体的分配为1：2对4 ：1为野生型酶。相对于野生型酶，A737V的核酸外切酶活性在单链DNA上没有变化。但是，在核酸外切酶和聚合酶活性位点之间分配双链体DNA的差异导致抗突变酶的核酸外切酶活性增强。 [参考：36]

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