Wilson Noah A, Abu-Shumays Robin, Gyarfas Brett, Wang Hongyun, Lieberman Kate R, Akeson Mark, Dunbar William B
Department of Computer Engineering, University of California, Santa Cruz, California, USA.
ACS Nano. 2009 Apr 28;3(4):995-1003. doi: 10.1021/nn9000897.
DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an alpha-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.
DNA聚合酶催化依赖模板的基因组复制。这些酶、其DNA底物的双链-单链连接以及与聚合酶活性位点中第一个模板碱基互补的脱氧核苷三磷酸（dNTP）之间形成高亲和力三元复合物，对这一过程至关重要。我们提出了一种单分子方法，用于以高时间分辨率迭代测量DNA-聚合酶复合物的组装，该方法利用了非共价连接在α-溶血素纳米孔中的单个DNA底物分子的有源电压控制。基于其离子电流特征诊断大肠杆菌DNA聚合酶I（KF）的Klenow片段的DNA结合状态，并以亚毫秒精度做出反应，以执行控制DNA底物与KF和dNTP接触的电压变化。对暴露时间的精确控制使得能够在与KF结合速率叠加的时间尺度上测量DNA-KF复合物的组装。在几秒钟内对单个连接的DNA分子进行了数百次测量，并且在单个实验中可以连接数十个分子。这种方法允许在单分子水平上对DNA和RNA加工酶及其底物之间复合物的组装进行具有统计学稳健性的分析。
