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首页> 外文期刊>Biochemistry >Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase.
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Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase.

机译：使用2-氨基嘌呤荧光检测噬菌体T4 DNA聚合酶掺入核苷酸过程中模板链中的碱基堆积。

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摘要

The fluorescence of the base analogue 2-aminopurine (2AP) was used to detect physical changes in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Fluorescent enzyme-DNA complexes were formed with 2AP placed in the template strand opposite the primer terminus (the n position) and placed one template position 5' to the primer terminus (the n + 1 position). The fluorescence enhancement for 2AP at the n position was shown to be due to formation of the editing complex, which indicates that the 2AP-T terminal base pair is recognized primarily as a mismatch. 2AP fluorescence at the n + 1 position, however, was a reporter for DNA interactions in the polymerase active center that induce intrastrand base unstacking. T4 DNA polymerase produced base unstacking at the n + 1 position following formation of the phosphodiester bond. Thus, the increase in fluorescence intensity for 2AP at the n + 1 position could be used to measure the nucleotide incorporation rate in primer extension reactions in which 2AP was placed initially at the n + 2 position. Primer extension occurred at the rate of about 314 s(-1). The amount of base unstacking at the template n + 1 position was sensitive to the local DNA sequence. More base unstacking was detected for DNA substrates with an A-T base pair at the primer terminus compared to C-G or G-C base pairs. Since proofreading is also increased by A-T base pairs compared to G-C base pairs at the primer terminus, we propose that base unstacking may provide an opportunity for the DNA polymerase to reexamine the primer terminus.
机译：碱基类似物2-氨基嘌呤（2AP）的荧光用于检测噬菌体T4 DNA聚合酶掺入核苷酸过程中模板链的物理变化。荧光酶-DNA复合物是通过将2AP放置在与引物末端相对的模板链（n位置）中，并在引物末端5'的一个模板位置（n + 1位置）形成的。显示了在n位置2AP的荧光增强是由于编辑复合物的形成，这表明2AP-T末端碱基对主要被识别为错配。然而，在n + 1位置的2AP荧光是聚合酶活性中心中引起链内碱基解堆积的DNA相互作用的报告基因。 T4 DNA聚合酶在形成磷酸二酯键后在n +1位产生碱基解堆。因此，在n + 1位置2AP的荧光强度增加可用于测量引物延伸反应中的核苷酸掺入率，其中2AP最初放置在n + 2位置。引物延伸发生的速度约为314 s（-1）。模板n + 1位置的碱基堆积量对局部DNA序列敏感。与C-G或G-C碱基对相比，在引物末端具有A-T碱基对的DNA底物检测到更多的碱基解叠。由于与引物末端处的G-C碱基对相比，A-T碱基对也增加了校对，因此我们建议碱基解叠可能为DNA聚合酶提供重新检查引物末端的机会。

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