Dohoney K M, Gelles J
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Nature. 2001 Jan 18;409(6818):370-4. doi: 10.1038/35053124.
Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.
大肠杆菌中重组DNA修复的主要途径需要RecBCD蛋白——一种异源三聚体、由ATP驱动的DNA转运运动酶。RecBCD将高度连续且异常快速的解旋酶（DNA解链）活性与链特异性核酸酶（DNA切割）活性结合在一起（参考文献1、2及其中的参考文献）。对DNA序列“chi”（5'-GCTGGTGG-3'）的识别会改变DNA切割的极性，并在体内刺激附近序列的重组。在这里，我们将微观聚苯乙烯珠附着到生物素标记的RecD蛋白亚基上，并使用系留颗粒光学显微镜观察单个RecBCD分子的转运（在2Hz时精度高达约30nm），并研究chi改变酶活性的机制。观察到的转运是单向的，每个分子以与群体平均DNA解链速率相对应的恒定速度移动。这些观察结果对可能的运动机制施加了严格限制。在chi处珠子释放可以忽略不计，这表明chi处的活性改变并不像先前提出的那样需要从酶中弹出RecD亚基；这种改变可能通过一种不同寻常的、纯粹的构象转换机制发生。
