Division of Biology, Indian Institute of Science Education and Research, Pune 411008, India.
Nucleic Acids Res. 2020 Mar 18;48(5):2594-2603. doi: 10.1093/nar/gkaa023.
Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a β-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.
参与核酸交易的酶通常具有解旋酶样 ATP 酶来协调和驱动其功能活动，但我们对其协调的机制细节的理解是有限的。例如，噬菌体防御系统 Type ISP 限制-修饰酶对 DNA 的切割需要两个这样的酶的收敛，这些酶在 Superfamily 2 ATPases 的驱动下在 DNA 上主动易位。当酶识别 DNA 靶序列时，ATP 酶被激活。在这里，我们表明激活是一个两步过程，即甲基转移酶和靶识别结构域识别靶序列后对 ATP 酶的部分刺激，以及另外需要 DNA 与 ATP 酶结构域相互作用的完全刺激。突变分析显示，ATP 酶的 β-发夹环和 motif V 将 DNA 易位与 ATP 水解偶联。环的缺失抑制了易位，而 motif V 的突变则降低了易位的速率。这两种突变都抑制了酶的双链 (ds) DNA 切割活性。然而，一个易位 motif V 突变在遇到易位的野生型酶时会切割 dsDNA。基于这些结果，我们得出结论，ATP 酶驱动的易位不仅将两个核酸酶空间上拉近以催化 dsDNA 断裂，而且易位的速率也会影响 dsDNA 的切割。
