Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility

Abstract

Homing endonucleases confer mobility to their host intervening sequence, either an intron or intein, by catalyzing a highly specific double-strand break in a cognate allele lacking the intervening sequence. These proteins are characterized by their ability to bind long DNA target sites (14-40 bp) and their tolerance of minor sequence changes in these sites. A wealth of biochemical and structural data has been generated for these enzymes over the past few years. Herein we review our current understanding of homing endonucleases, including their diversity and evolution, DNA-binding and catalytic mechanisms, and attempts to engineer them to bind novel DNA substrates.

Figure 1

Homing mechanisms of group I introns (left), inteins (center) and group II introns (right) in which the intervening sequence of gene X is duplicated in its cognate allele, gene X′. Mobile ORFs and encoded products are green; host gene exons and products are red; other nucleic acid sequence is black. E, endonuclease; M, maturase; RT, reverse-transcriptase; RNP, ribonucleoprotein.

Figure 2

Structures of LAGLIDADG family members. Endonuclease domains are blue with orange β-sheets showing DNA-binding saddle. Other intein domains are gray. (A) I-CreI homodimer. (B) I-CreI with DNA. (C) I-DmoI monomer. (D) PI-PfuI monomer. (E) PI-SceI monomer.

Figure 3

His-Cys box family. (A) I-PpoI homodimer bound to its DNA target site. Note ‘domain-swapped’ C-terminal tails that form much of the dimer interface. Zinc atoms are green. (B) Core metal-binding motifs in I-PpoI. Zinc ions are green; magnesium ion is purple. β-strands and α-helix of the conserved ββαMe motif are red and blue, respectively. (C) Primary sequence alignment of known His-Cys box family members including I-NjaI, I-NanI, I-NitI, I-DirI and I-PpoI. The C-terminal dimerization tail of I-PpoI and the highly conserved zinc-binding and active sight residues are highlighted.

Figure 4

DNA-binding by homing and restriction endonucleases. (A) Summary of actively cleaved target sites containing single base changes. I-CreI and I-PpoI recognize degenerate palindromes; EcoRV recognizes a strict palindrome. Palindromic bases are boxed. The homing endonucleases I-CreI and I-PpoI are tolerant of minor changes in their homing site as shown. The restriction endonuclease EcoRV is intolerant of any changes to its restriction site. Generally, the level of tolerance correlates inversely to number of specific protein–DNA contacts. Mutant homing site data taken from Argast et al. (114) and restriction site data from Pingoud and Jeltsch (112). (B) Base-specific contacts made by each endonuclease. Major groove contacts are drawn to the top of the base; minor groove contacts are drawn to the bottom of the base. Hydrogen bonds are represented as solid lines; other interactions are dashed lines.

Figure 5

Endonuclease cleavage transition states. In all figures, the blue lines represent bonds in transition. Red lines represent the breaking bond of the DNA backbone. (A) Phosphodiester bond cleavage requires three chemical entities: a general base to activate the nucleophile, a Lewis acid to stabilize the pentacoordinate phosphoanion transition state and a general acid to protonate the 3′ leaving group. Water molecules usually function as nucleophiles and often as general acids. Divalent metal cations function as Lewis acids, can help lower the pK_a of associated waters and position the nucleophile. Other residues act as general bases and/or to position waters and metal ions. (B) BglII uses a single metal in its mechanism. The magnesium functions to lower pK_a of nucleophile and stabilize the transition state. The general base appears to be Gln95; the general acid has not been conclusively identified. (C) In BamHI, two magnesium ions aid in catalysis by positioning and lowering the pK_a of the nucleophile and proton donor and stabilizing the phosphoanion transition state. Glu113 is the general base and water is the general acid. (D) I-PpoI, like BglII, uses a single-metal mechanism. In I-PpoI, the metal stabilizes the transition state and activates the general acid, an associated water molecule. It does not appear to participate in positioning and/or activating the nucleophile. The general base is His98. (E) I-CreI uses a two-metal mechanism, the central metal being shared by two separate active sites. The metals position and lower the pK_a of the nucleophile, stabilize the transition state and stabilize the 3′ oxygen leaving group. The general base and acid appear to be within a well-ordered solvent network (positioned by peripheral residues and the first metal ion); for clarity, only three of these water molecules—other than the nucleophile—are drawn.

Figure 6

Structural alignment of LAGLIDADG active sites. View is top-down through protein body to active site residues and DNA below. In all three diagrams, subunits of the homodimer I-CreI are blue and purple and are labeled as I-CreI and I-CreI′. The DNA backbone (gray) and the three divalent ions (black) from I-CreI–DNA co-crystal structures are shown for reference. Note the enormous divergence in both amino acid identity and position at the periphery of the active sites. (A) I-DmoI over I-CreI. (B) PI-PfuI over I-CreI. (C) PI-SceI over I-CreI. Alignments generated by superposition of LAGLIDADG α-helices.

Figure 7

Diagram of the I-CreI active site (A) in the presence of calcium (non-cleaved DNA substrate) and (B) in the presence of magnesium (cleaved DNA substrate). DNA is red, metal ions are green, protein side chains are black. Waters are light blue; distances between waters are shown in Å by the associated blue lines representing the hydrogen-bonding network. The nucleophilic water is purple. Note that the protein makes no direct contact to the nucleophile, the scissile phosphate (red outlined in black), or the 3′ leaving group and that the solvent network extends from the nucleophile to the leaving group.