Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase
Stopped-flow fluorescence study of precatalytic primer strand base-unstacking transitions in the exonuclease cleft of bacteriophage T4 DNA polymerase
复制标题
DOI：
10.1021/bi9800754
复制
发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Beechem, JM
中科院分区：
文献类型：
--
作者：
Otto, MR;Bloom, LB;Beechem, JM
DNA 聚合酶是结合引物模板 DNA 并随后延伸或切除引物链上末端核苷酸的复杂酶。在本研究中，停流荧光各向异性结合测定与位于 3' 引物末端的荧光腺嘌呤类似物（2-氨基嘌呤）的实时测量相结合。使用这种组合方法，检查了与蛋白质结合、引物末端解堆积以及噬菌体 T4 (T4 pol) 的 3'→5' 核酸外切酶进行的碱基切除相关的确切时间过程。 T4 pol 结合和解离动力学被发现遵循简单的动力学，单链引物和双链引物模板（100 muM Mg2+）具有相同的结合速率（k(on) = 4.6 x 10(8) M-1 s(-1)）和解离速率（k(off) = 9.3 s(-1)）。尽管 T4 pol-DNA 结合和解离的时间过程遵循简单的动力学，但在次优 Mg2+ 浓度（例如 100 μM）下，双链引物模板中引物末端的碱基解堆积反应观察到非一级 S 形动力学。观察到的碱基解堆积的 S 形动力学表明 T4 pol 是一种滞后酶 [Frieden, C. (1970) J. Biol.化学。 245, 5788-5799] 并且必须以两种 DNA 结合构象存在，这两种构象在碱基解堆积特性上差异很大。在引物-模板结合和解堆积转变开始之间观察到 10 ms 的 Mg2+ 依赖性时间延迟，在添加 100 muM Mg2+ 后 22 +/- 1 ms 时解堆积转变完成 50%。在滞后滞后之后，解决了 130 s(-1) 的简单一级引物末端解堆积速率，该速率与蛋白质和 Mg2+ 浓度无关。对于单链引物的处理，所有动力学复杂性都会丢失，并且 T4 pol 结合和引物末端碱基解堆积动力学可以叠加。这些数据表明，T4 pol 对双链引物模板 DNA 的动力学处理比单链引物复杂得多，并且表明聚合酶和核酸外切酶位点之间的内在“转换速率”可能比之前提出的要快得多。
DNA polymerases are complex enzymes which bind primer-template DNA and subsequently either extend or excise the terminal nucleotide on the primer strand. In this study, a stopped-flow fluorescence anisotropy binding assay is combined with real-time measurements of a fluorescent adenine analogue (2-aminopurine) located at the 3'-primer terminus. Using this combined approach, the exact time course associated with protein binding, primer terminus unstacking, and base excision by the 3' --> 5' exonuclease of bacteriophage T4 (T4 pol) was examined. T4 pol binding and dissociation kinetics were found to obey simple kinetics, with identical on rates (k(on) = 4.6 x 10(8) M-1 s(-1)) and off rates (k(off) = 9.3 s(-1)) for both single-stranded primers and double-stranded primer-templates (at 100 mu M Mg2+). Although the time course for T4 pol-DNA association and dissociation obeyed simple kinetics, at suboptimal Mg2+ concentrations (e.g., 100 mu M), non-first-order sigmoidal kinetics were observed for the base-unstacking reaction of the primer terminus in double-stranded primer-templates. The observed sigmoidal kinetics for base unstacking demonstrate that T4 pol is a hysteretic enzyme [Frieden, C. (1970) J. Biol. Chem. 245, 5788-5799] and must exist in two DNA bound conformations which differ greatly in base-unstacking properties. A Mg2+-dependent time lag of 10 ms is observed between primer-template binding and the beginning of the unstacking transition, which is 50% complete at 22 +/- 1 ms after addition of 100 mu M Mg2+. Following the hysteretic lag, a simple first-order primer terminus unstacking rate of 130 s(-1) is resolved, which is protein and Mg2+ concentration-independent. For the processing of single-stranded primers, all kinetic complexity is lost, and T4 pol binding and primer end base-unstacking kinetics can be superimposed. These data reveal that the kinetic processing of double-stranded primer-template DNA by T4 pol is much more complex than that of single-stranded primers, and suggest that the intrinsic "switching rate" between the polymerase and exonuclease sites may be much faster than previously proposed.