Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli.
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.
Wang
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J
Department of Botany and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78713, USA.
Kim
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Yuan
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Herrin
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D017665:Hydroxyl Radical; D008274:Magnesium / Q000378:metabolism; D008745:Methylation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding
D017931:DNA Primers; D017665:Hydroxyl Radical; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; D008274:Magnesium