Tóth Júlia, Bollins Jack, Szczelkun Mark D
DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
DNA-Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK
Nucleic Acids Res. 2015 Dec 15;43(22):10870-81. doi: 10.1093/nar/gkv1154. Epub 2015 Nov 3.
DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.
III型限制酶切割DNA需要识别位点之间通过热驱动的一维扩散促进的长距离蛋白质通讯。这种“DNA滑动”由类解旋酶结构域催化的多个ATP水解引发。使用短寡双链底物观察到两个不同的ATP酶阶段；约10个ATP的快速消耗与蛋白质构象转换相关，随后是较慢的阶段，其持续时间由从识别位点解离的速率决定。在这里，我们表明第二个ATP酶阶段是可变的，并且只有当DNA末端靠近识别位点时才能观察到。在DNA上，位点距离末端更远时，观察到与构象转换相关的单个ATP酶阶段，随后的位点解离几乎不需要或不需要进一步的ATP水解。整体DNA解离动力学（包括位点释放、DNA滑动和通过DNA末端逃逸）不受第二阶段的影响。尽管这些数据简化了III型限制酶的ATP水解模式，但关于为什么要水解多个ATP来为DNA滑动做准备的问题仍然存在。
