Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI

Abstract

Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

Figure 1. Model of I-BmoI interactions with intronless and intron-containing substrates based on DNA footprinting experiments.

Shown is a schematic of the modular interaction of I-BmoI with the intronless thyA allele (upper), and the resultant changes to the target site upon intron insertion that generates the intron-containing allele (lower). Top- and bottom-strand nicking sites are indicated by open and filled triangles, respectively. The critical GC-2 base-pair is shown in enlarged bold-type font, and the intron insertion site (IS) is indicated by a vertical line with exon 2 downstream and either exon 1 or the intron (red) upstream.

Figure 2. Magnesium is the preferred divalent metal ion for efficient and specific cleavage by I-BmoI.

A. Representative gel images of time-point cleavage assays with I-BmoI performed on supercoiled substrate containing the intronless thyA target site. Reactions contained increasing concentrations of MgCl₂, CaCl₂, CuCl₂, MnCl₂, NiCl₂, and ZnCl₂. Lanes that lack I-BmoI (-) have 10 mM metal (1 mM for ZnCl₂), and nicked (N), linear (L), and circular (C) plasmid forms are indicated to the right of each gel image. B. Representative gel images of I-BmoI cleavage assays performed on supercoiled substrate containing a mutation at the critical GC-2 basepair (G-2T). Reactions were performed in the presence of MgCl₂ or MnCl₂.

Figure 3. Limiting divalent metal ion has a greater effect on second strand than first strand nicking.

Shown are representative images of time-course cleavage assays with supercoiled substrate containing the intronless thyA target site and I-BmoI in A. 0.5 mM MgCl₂ and B. 10 mM MgCl₂, as well as progress curves for each condition. Circular substrate (C), nicked intermediate (N) and linear product (L) are indicated on the gel images. Individual data points from three independent replicates are shown in the progress curves, and the solid continuous lines are the best fit of the data to equations 1 and 2. C. Plot of the log₁₀ k₁ - log₁₀ k₂ value for MgCl₂ concentrations tested.

Figure 4. Magnesium concentrations reveal three distinct classes of cleavage site substitutions.

Cleavage assays with I-BmoI were conducted on supercoiled plasmid substrates containing substitutions at positions -6 to -1 in the presence of 2 mM or 10 mM MgCl₂ (see also Table 2). Mutant substrates were arranged into three classes. (A) Substrates that showed a phenotype similar to wild-type intronless thyA substrate (class I); (B) substrates that demonstrated poor cleavage in 2 mM MgCl₂ and rescued cleavage in 10 mM MgCl₂ (class II); (C) substrates with significantly reduced or no cleavage (class III). Shown are representative gel images of time-course cleavage assays, where the second lane from the left contains unreacted plasmid substrate (-). Nicked (N), linear (L), and circular (C) plasmid forms indicated to the right. Beneath each gel image is a graphical representation of reaction progress over time in 2 mM and 10 mM MgCl₂ using dashed and solid lines, respectively. Data points representing three independent experiments for class I and II, and two experiments for class III are shown.

Figure 5. The GC-2 base pair is required for minor-groove distortion near the bottom-strand nick site.

(A) Illustration of base pairs -6 to +7 of the intronless (In-), intron-containing (In+), and mutant 74-mer substrates used for in-gel 1,10-phenanthroline copper (OP-Cu) footprinting reactions. First- and second-strand nicking sites are indicated by open and filled triangles, respectively. (B) Representative denaturing gel image of OP-Cu footprinting reactions on bottom-strand labeled In-, In+, and mutant substrates. Nicked products at -4 and minor groove distortions at positions -2 and -1 are indicated using filled triangles, asterisks, and greater-than symbols, respectively. Sequence upstream of the intron insertion site (IS) varies whether the intronless or intron-containing substrate was used as a template. (C) Graphical representation of the minor groove sensitivity to OP-Cu at positions -2 (asterisk, blue) and -1 (greater than symbol, purple) for In-, In+, and mutant substrates, expressed as the ratio of normalized phosphorimager units of the upper complex (UC) and unbound substrate (UNB).

Figure 6. Model of I-BmoI GIY-YIG domain interactions with substrate.

(A) Cartoon representation of the I-BmoI GIY-YIG domain homology model (gray) aligned with the solution structure of related GIY-YIG restriction enzyme Eco29kI (3MX1, blue) . The side chains of the conserved and catalytically relevant residues Y6, Y17, R27, H31, E74, and N87 of I-BmoI are shown in black, and equivalent Eco29kI residues in orange (Y49, Y76, R104, H108, E142, and N154). The position of divalent metal coordinated by Eco29kI is represented by the red sphere. (B) Cartoon representation of the I-BmoI homology model aligned with a segment of the DNA from the substrate-bound Eco29kI structure (3N1C,-4C to +5C). The top strand of the Eco29kI substrate is labeled according to the nucleotides of the I-BmoI intronless allele (-8G to +1A). The phosphate of the bottom strand nick site is highlighted in blue, the bottom strand distortions at positions -2 and -1 are shown in green, and the -2GC base pair is shown as a stick model in red. (C) Surface representation of the GIY-YIG domain for the model shown in panel (B). (D) Similar to panel (C), with a 90° rotation of the model on the horizontal axis, and a 180° rotation around the vertical axis.