Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA Polymerases
Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA Polymerases
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DOI：
10.1074/jbc.m203315200
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发表时间：
2002-10-25
影响因子：
4.8
通讯作者：
Reha-Krantz, LJ
中科院分区：
文献类型：
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作者：
da Silva, EF;Mandal, SS;Reha-Krantz, LJ
野生型和突变型 T4 DNA 聚合酶区分碱基类似物 2-氨基嘌呤 (2AP) 的利用的能力和 2AP 的荧光被用来确定 DNA 聚合酶如何区分正确和错误的核苷酸。由于 T4 DNA 聚合酶在单周转条件下掺入 dTMP 相对 2AP，因此可以直接比较掺入 dTMP 相对模板 2AP 的动力学参数与掺入 dTMP 相对模板 A 的参数，而不会出现酶解离的复杂情况。检测到的最显着差异是 dTTP 的 K-d，掺入 dTMP 相对模板 2AP（类似于 367 mum）的 K-d 比掺入 dTMP 相对模板 A（类似于 31 mum）高 10 倍。相反，dTMP掺入率仅降低约2倍，从模板A的约318s(-1)减少到模板2AP的约165s(-1)。歧视是由于初始核苷酸结合步骤中的高选择性。 T4 DNA 聚合酶与模板位置具有 2AP 的 DNA 结合，诱导形成核苷酸结合袋，该核苷酸结合袋经过预成形以结合 dTTP 并排除其他核苷酸。如果核苷酸结合受到阻碍，校对途径的启动将充当错误避免机制，以防止掺入不正确的核苷酸。
The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K-d for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (similar to367 mum) than for incorporation of dTMP opposite template A (similar to31 mum). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.