Purohit Vandana, Grindley Nigel D F, Joyce Catherine M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 2003 Sep 2;42(34):10200-11. doi: 10.1021/bi0341206.
We have investigated conformational transitions in the Klenow fragment polymerase reaction by stopped-flow fluorescence using DNA substrates containing the fluorescent reporter 2-aminopurine (2-AP) on the template strand, either at the templating position opposite the incoming nucleotide (designated the 0 position) or 5' to the templating base (the +1 position). By using both deoxy- and dideoxy-terminated primers, we were able to distinguish steps that accompany ternary complex formation from those that occur during nucleotide incorporation. The fluorescence changes revealed two extremely rapid steps that occur early in the pathway for correct nucleotide incorporation. The first, detectable with the 2-AP reporter at the 0 position, occurs within the first few milliseconds and is associated with dNTP binding. This is followed by a rapid step involving relative movement of the +1 base, detectable when the 2-AP reporter is at the +1 position. Finally, when the primer had a 3'-OH, a fluorescence decrease with a rate equal to the rate of nucleotide incorporation was observed with both 0 and +1 position reporters. When the primer was dideoxy-terminated, the only change observed at the rate expected for nucleotide incorporation had a very small amplitude, suggesting that the rate-limiting conformational change does not produce a large fluorescence change, and is therefore unlikely to involve a significant change in the environment of the fluorophore. Fluorescence changes observed during misincorporation were substantially different from those observed during correct nucleotide incorporation, implying that the conformations adopted during correct and incorrect nucleotide incorporation are distinct.
我们通过停流荧光法研究了Klenow片段聚合酶反应中的构象转变，所用的DNA底物在模板链上含有荧光报告基团2-氨基嘌呤（2-AP），其位置要么在与进入的核苷酸相对的模板位置（称为0位置），要么在模板碱基的5'端（+1位置）。通过使用脱氧和双脱氧终止的引物，我们能够区分三元复合物形成过程中的步骤与核苷酸掺入过程中发生的步骤。荧光变化揭示了在正确核苷酸掺入途径早期发生的两个极其快速的步骤。第一个步骤，用位于0位置的2-AP报告基团可检测到，发生在最初几毫秒内，与dNTP结合有关。接着是一个涉及+1碱基相对移动的快速步骤，当2-AP报告基团位于+1位置时可检测到。最后，当引物具有3'-OH时，在0和+1位置报告基团处均观察到荧光下降，其速率等于核苷酸掺入速率。当引物是双脱氧终止时，在预期的核苷酸掺入速率下观察到的唯一变化幅度非常小，这表明限速构象变化不会产生大的荧光变化，因此不太可能涉及荧光团环境的显著变化。错配掺入过程中观察到的荧光变化与正确核苷酸掺入过程中观察到的荧光变化有很大不同，这意味着正确和错误核苷酸掺入过程中所采用的构象是不同的。
