PURIFICATION, BIOCHEMICAL-CHARACTERIZATION AND PROTEIN-DNA INTERACTIONS OF THE I-CREI ENDONUCLEASE PRODUCED IN ESCHERICHIA-COLI

I-Crel is a member of the LAGLI-DADG family of homing nucleases; howev er, unlike most members of this family it contains only a single copy of this signature motif, I-Crel was over-expressed in Escherichia coli , and a simple purification protocol developed that gave reasonably pu re protein in high yield, Size-exclusion chromatography and chemical c ross-linking indicated that the protein is a dimer in solution, DNA cl eavage by I-Crel was absolutely dependent on Mg2+ (or Mn2+), and was i nhibited by monovalent cations, I-Crel displayed a surprisingly high t emperature optimum (>50 degrees C), with full activity occurring even at 70 degrees C. Interestingly, SDS was needed for efficient release o f the cleavage products from the protein, indicating formation of very stable DNA-protein complexes, In contrast to these robust characteris tics, purified I-Crel was unstable; however, it could be stabilized by the addition of either target or non-target DNA, Mobility shift assay s revealed that I-Crel binds to DNA in the absence of Mg2+ Hydroxyl ra dical footprinting showed that I-Crel strongly protected the backbone of a continuous stretch of at least 12 nt on each strand that were shi fted, relative to each other, by 2 bp in the 3' direction, Methylation protection and interference analyses were also performed, and togethe r with the hydroxyl radical footprinting, indicate that I-Crel binds i n both the major and minor grooves of its target DNA.