Purification, Biochemical Characterization and Protein-DNA Interactions of the I-CreI Endonuclease Produced in Escherichia Coli

Abstract

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.

Characterization of Mycobacterium leprae RecA Intein, a LAGLIDADG Homing Endonuclease, Reveals a Unique Mode of DNA Binding, Helical Distortion, and Cleavage Compared with a Canonical LAGLIDADG Homing Endonuclease

“…5D) or Mg 2ϩ (data not shown). The maximum catalytic activity of PI-MleI observed in the presence of 1-5 mM Mn 2ϩ or Mg 2ϩ is comparable with that of other homing endonucleases (12,41,42). Although a majority of known homing endonucleases requires 3-5 mM divalent cations for their optimal activity, PI-TfuI (Thermococcus fumicolans) displays its maximum catalytic activity in the presence of 25 mM Mn 2ϩ (43).…”

“…Effect of Ionic Strength on DNA Cleavage by PI-MleI-The assay buffer contained 25 mM Tris-HCl and 3 mM MnCl 2 or MgCl 2 , which is comparable with standard conditions used in studies with other homing endonucleases (12,13,41). We therefore wished to test the effect of increasing ionic strength on PI-MleI endonuclease activity.…”

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

“…[3][4][5] Several lines of evidence suggest that a variety of divalent cations can support cleavage reactions by HEases, but they do not influence DNA-binding. [10][11][12] Most HEases prefer oxophilic Mg 2þ , some use thiophilic Mn 2þ , whereas others can use both for optimal catalysis. 10,11 Cocrystal structures of several LAGLI-DADG endonucleases including I-CreI, 13 I-MsoI, 14 and I-SceI 15 indicated the presence of three divalent cations, coordinated by a pair of conserved aspartate residues at the active site.…”

“…[10][11][12] Most HEases prefer oxophilic Mg 2þ , some use thiophilic Mn 2þ , whereas others can use both for optimal catalysis. 10,11 Cocrystal structures of several LAGLI-DADG endonucleases including I-CreI, 13 I-MsoI, 14 and I-SceI 15 indicated the presence of three divalent cations, coordinated by a pair of conserved aspartate residues at the active site. On the other hand, cocrystal structures of I-AniI 16 and PI-SceI 17 showed the presence of two divalent cations with shared metal being not visible.…”

Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA

“…Homing endonucleases with only one such motif, such as I-CreI (Wang et al, 1997), function as homodimers. In contrast, larger HEs containing two motifs, such as I-SceI (Jacquier & Dujon, 1985) or I-Dmo-I (Dalgaard et al, 1993), are single-chain proteins.…”