Sexton D J, Carver T E, Berdis A J, Benkovic S J
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
J Biol Chem. 1996 Nov 8;271(45):28045-51. doi: 10.1074/jbc.271.45.28045.
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.
T4 DNA聚合酶全酶由与滑动夹（45蛋白）复合的聚合酶组成，滑动夹由ATP依赖的夹加载器（44/62复合物）加载到DNA上。本文描述了一种使用45蛋白的荧光标记半胱氨酸突变体直接研究全酶组装机制的新方法。该蛋白功能未改变，但与全酶的其他成分相互作用时，探针荧光强度发生了显著变化。这些荧光变化为ATP水解在全酶组装中的作用提供了见解。使用ATP或不可水解的ATP类似物腺苷5'-O-(3-硫代磷酸酯)，全酶组装过程中的事件被确定为依赖或独立于ATP水解。提出了一种全酶组装机制，其中44/62复合物以依赖ATP但不需要ATP水解的方式介导45蛋白与DNA的结合。ATP水解后，44/62复合物触发45蛋白的构象变化，这可能归因于夹加载到DNA上。
