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.

中文翻译：

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DNA介导的ISP型限制性修饰酶的ATPase，转位酶和核酸酶活性的偶联。

参与核酸交易的酶通常具有类似解旋酶的ATPase来协调和驱动其功能活动，但我们对它们的协调机制细节的了解有限。例如，通过抗噬菌体防御系统类型ISP限制性修饰酶进行的DNA切割需要两种此类酶的融合，它们在由超家族2 ATP酶驱动的DNA上活跃易位。当该酶识别DNA靶序列时，ATPase被激活。在这里，我们表明激活是甲基转移酶和靶标识别域识别靶序列后进行部分ATPase刺激的两阶段过程，完全刺激还需要DNA与ATPase域相互作用。诱变表明，β-发夹环和ATPase的基序V将DNA易位与ATP水解耦合。环的删除抑制了易位，而基序V的突变减慢了易位的速率。两种突变均抑制了该酶的双链（ds）DNA切割活性。然而，易位基序V突变体在遇到易位野生型酶时切割了dsDNA。根据这些结果，我们得出结论，由ATPase驱动的易位不仅使两个核酸酶在空间上接近催化dsDNA断裂，而且易位速率影响dsDNA裂解。然而，易位基序V突变体在遇到易位野生型酶时切割了dsDNA。根据这些结果，我们得出结论，由ATPase驱动的易位不仅使两个核酸酶在空间上接近催化dsDNA断裂，而且易位速率影响dsDNA裂解。然而，易位基序V突变体在遇到易位野生型酶时切割了dsDNA。根据这些结果，我们得出结论，由ATPase驱动的易位不仅使两个核酸酶在空间上接近催化dsDNA断裂，而且易位速率影响dsDNA裂解。