EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities.
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.
Institute of Cell and Molecular Biology, Darwin Building, King's Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
G P
GP
Davies
L M
LM
Powell
J L
JL
Webb
L P
LP
Cooper
N E
NE
Murray
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015183:Restriction Mapping
D017931:DNA Primers; D004269:DNA, Bacterial; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; D000251:Adenosine Triphosphatases