Nucleic Acids Research, Vol 27, Issue 21 4200-4207, Copyright © 1999 by Oxford University Press
XJ Zhang and DA Julin
The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in
previous work to have two domains: an N-terminal 100 kDa domain with
ATP-dependent helicase activity, and a C-terminal 30 kDa domain. The 30 kDa
domain had nuclease activity when linked to a heterologous DNA binding
protein, but by itself it appeared unable to bind DNA and lacked detectable
nuclease activity. We have expressed and isolated this 30 kDa domain,
called RecB(N), and show that it does have nuclease activity detectable at
high protein concentration in the presence of polyethylene glycol, added as
a molecular crowding agent. The activity is undetectable in a mutant
RecB(N)protein in which an aspartate residue has been changed to alanine.
Structural analysis of the wild- type and mutant RecB(N)proteins by second
derivative absorbance and circular dichroism spectroscopy indicates that
both are folded proteins with very similar secondary and tertiary
structures. The results show that the Asp-->Ala mutation has not caused
a significant structural change in the isolated domain and they support the
conclusion that the C-terminal domain of RecB has the sole nuclease active
site of RecBCD.
ARTICLES
Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
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