Nucleic Acids Research, Vol 25, Issue 18 3687-3692, Copyright © 1997 by Oxford University Press
H Kong and CL Smith
The BcgI restriction-modification system consists of two subunits, A and B.
It is a bifunctional protein complex which can cleave or methylate DNA. The
regulation of these competing activities is determined by the DNA
substrates and cofactors. BcgI is an active endonuclease and a poor
methyltransferase on unmodified DNA substrates. In contrast, BcgI is an
active methyltransferase and an inactive endonuclease on hemimethylated DNA
substrates. The cleavage and methylation reactions share cofactors. While
BcgI requires Mg2+and S - adenosyl methionine (AdoMet) for DNA cleavage,
its methylation reaction requires only AdoMet and yet is significantly
stimulated by Mg2+. Site- directed mutagenesis was carried out to
investigate the relationship between AdoMet binding and BcgI DNA
cleavage/methylation activities. Most substitutions of conserved residues
forming the AdoMet binding pocket in the A subunit abolished both
methylation and cleavage activities, indicating that AdoMet binding is an
early common step required for both cleavage and methylation. However, one
mutation (Y439A) abolished only the methylation activity, not the DNA
cleavage activity. This mutant protein was purified and its methylation,
cleavage and AdoMet binding activities were tested in vitro . BcgI- Y439A
had no detectable methylation activity, but it retained 40% of the AdoMet
binding and DNA cleavage activities.
ARTICLES
Substrate DNA and cofactor regulate the activities of a multi- functional restriction-modification enzyme, BcgI
New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA. kong@neb.com
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