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Abstract
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.
Highlights
IntroductionHelicases and translocases are the primary motors facilitating nucleic acids transactions in a cell [1]
It has been shown that at least 23 bp upstream to the target sequence is required for LlaBIII to initiate translocation along the DNA (Figure 1A) [4]. This prompted us to ask if the binding of the target sequence to the target recognition domain (TRD) and MTase is sufficient to stimulate the ATPase or, like in the case of translocation initiation, if a DNA long enough to interact with the ATPase is required? It has been demonstrated in the case of some SF2 helicases that motifs III and V transduce the conformational changes arising from ATP hydrolysis to DNA translocation [11–18]
The study described here aimed at understanding the mechanism of stimulation of the ATPase activity and its coupling to the other functional activities of the Type ISP RM enzyme
Summary
Helicases and translocases are the primary motors facilitating nucleic acids transactions in a cell [1]. They hydrolyze nucleoside triphosphate and use the chemical energy to perform mechanical work. Helicases unwind double stranded (ds) DNA and directionally move (translocate) along the unwound single stranded DNA, while translocases translocate along dsDNA [1]. The detailed mechanism of how the different domains are functionally coupled to the motor in many of these molecular machines is not well understood [1,2]. We have studied the ATPase activity of the Type ISP restriction-modification (RM) enzyme leading to translocation initiation, and gain insights into the importance of enzyme translocation for its single-strand nicking and double-strand DNA cleavage activities
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