Mutations in the Res Subunit of the Eco PI Restriction Enzyme that Affect ATP-dependent Reactions

Author: Saha S.   Rao D.N.  

Publisher: Academic Press

ISSN: 0022-2836

Source: Journal of Molecular Biology, Vol.269, Iss.3, 1997-06, pp. : 342-354

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Abstract

The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by Eco PI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.Eco PIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in Eco PI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that Eco PI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes.