Nucleic Acids Research, Vol 27, Issue 13 2638-2643, Copyright © 1999 by Oxford University Press
P Janscak, U Sandmeier and TA Bickle
Type I restriction enzymes bind to specific DNA sequences but subsequently
translocate non-specific DNA past the complex in a reaction coupled to ATP
hydrolysis and cleave DNA at any barrier that can halt the translocation
process. The restriction subunit of these enzymes, HsdR, contains a cluster
of seven amino acid sequence motifs typical of helicase superfamily II,
that are believed to be relevant to the ATP-dependent DNA translocation.
Alignment of all available HsdR sequences reveals an additional conserved
region at the protein N- terminus with a consensus sequence reminiscent of
the P-D.(D/E)-X-K catalytic motif of many type II restriction enzymes. To
investigate the role of these conserved residues, we have produced mutants
of the type IB restriction enzyme Eco AI. We have found that single alanine
substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit
abolished the enzyme's restriction activity but had no effect on its ATPase
and DNA translocation activities, suggesting that these residues are part
of the active site for DNA cleavage.
ARTICLES
Single amino acid substitutions in the HsdR subunit of the type IB restriction enzyme EcoAI uncouple the DNA translocation and DNA cleavage activities of the enzyme
Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056, Switzerland.
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