Nucleic Acids Research, Vol 26, Issue 21 4828-4836, Copyright © 1998 by Oxford University Press
GP Davies, LM Powell, JL Webb, LP Cooper and NE Murray
For type I restriction systems, recently determined nucleotide sequences
predict conserved amino acids in the subunit that is essential for
restriction but not modification (HsdR). The conserved sequences emphasize
motifs characteristic of the DEAD-box family of proteins which comprises
putative helicases, and they identify a new candidate for motif IV. We
provide evidence based on an analysis of Eco KI which supports both the
relevance of DEAD-box motifs to the mechanism of restriction and the new
definition of motif IV. Amino acid substitutions within the newly
identified motif IV and those in six other previously identified DEAD-box
motifs, but not in the original motif IV, confer restriction-deficient
phenotypes. We have examined the relevance of the DEAD-box motifs to the
restriction pathway by determining the steps permitted in vitro by the
defective enzymes resulting from amino acid substitutions in each of the
seven motifs. Eco KI purified from the seven restriction-deficient mutants
binds to an unmethylated target sequence and, in the presence of AdoMet,
responds to ATP by undergoing the conformational change essential for the
pathway of events leading to DNA cleavage. The seven enzymes have little or
no ATPase activity and no endonuclease activity, but they retain the
ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA
strand could therefore be an essential early step in the restriction
pathway, facilitating the ATP-dependent translocation of DNA, particularly
if this involves DNA helicase activity.
ARTICLES
EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities
Institute of Cell and Molecular Biology, Darwin Building, King's Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
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