Nucleic Acids Research, Vol 25, Issue 19 3767-3776, Copyright © 1997 by Oxford University Press
J Wang, HH Kim, X Yuan and DL Herrin
I- CreI is a member of the LAGLI-DADG family of homing nucleases; however,
unlike most members of this family it contains only a single copy of this
signature motif. I- CreI was over-expressed in Escherichia coli, and a
simple purification protocol developed that gave reasonably pure protein in
high yield. Size-exclusion chromatography and chemical cross-linking
indicated that the protein is a dimer in solution. DNA cleavage by I- CreI
was absolutely dependent on Mg2+(or Mn2+), and was inhibited by monovalent
cations. I- CreI displayed a surprisingly high temperature optimum (>50
degrees C), with full activity occurring even at 70 degrees C.
Interestingly, SDS was needed for efficient release of the cleavage
products from the protein, indicating formation of very stable DNA-protein
complexes. In contrast to these robust characteristics, purified I- CreI
was unstable; however, it could be stabilized by the addition of either
target or non-target DNA. Mobility shift assays revealed that I- CreI binds
to DNA in the absence of Mg2+. Hydroxyl radical footprinting showed that I-
CreI strongly protected the backbone of a continuous stretch of at least 12
nt on each strand that were shifted, relative to each other, by 2 bp in the
3'direction. Methylation protection and interference analyses were also
performed, and together with the hydroxyl radical footprinting, indicate
that I- CreI binds in both the major and minor grooves of its target DNA.
ARTICLES
Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli
Department of Botany and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78713, USA.
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