Maine I P, Kodadek T
Department of Chemistry and Biochemistry, University of Texas at Austin 78712.
Biochem Biophys Res Commun. 1994 Feb 15;198(3):1070-7. doi: 10.1006/bbrc.1994.1152.
The structural nature of helicase-substrate complexes in the unwinding mode is difficult to study due to their transient nature. We report here a simple method to freeze a DNA helicase at a specific position. The method employs a sequence-specific DNA protein complex as a "roadblock" to helicase movement. The feasibility of the approach is demonstrated by trapping the dda protein of bacteriophage T4 upstream of a GAL4-DNA complex. The presence of the trapped helicase is demonstrated directly by protection of a nearby restriction site and indirectly by the inability of the helicase to recycle rapidly to unwind an unmodified substrate. The half-life of this frozen complex is approximately two minutes under the conditions employed. These results suggest that further study of this novel complex will prove fruitful in elucidating the properties of a DNA helicase in its unwinding mode. As a case in point, it is shown that the dda protein ceases to hydrolyze ATP while stalled, suggesting that nucleotide triphosphate hydrolysis is coupled to translocation for this enzyme.
由于解旋酶 - 底物复合物处于解旋模式时具有瞬时性，其结构性质很难研究。我们在此报告一种将DNA解旋酶冻结在特定位置的简单方法。该方法利用序列特异性DNA - 蛋白质复合物作为解旋酶移动的“路障”。通过在GAL4 - DNA复合物上游捕获噬菌体T4的dda蛋白，证明了该方法的可行性。通过保护附近的限制性酶切位点直接证明了捕获的解旋酶的存在，通过解旋酶无法快速循环以解旋未修饰的底物间接证明了这一点。在所采用的条件下，这种冻结复合物的半衰期约为两分钟。这些结果表明，对这种新型复合物的进一步研究将有助于阐明DNA解旋酶在其解旋模式下的性质。例如，结果表明dda蛋白在停滞时停止水解ATP，这表明该酶的三磷酸核苷酸水解与易位相偶联。
