Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

Abstract

Background: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.

Results: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme.

Conclusion: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.

Figure 1

Multiple sequence alignment of R.Eco29kI and related REases with the experimentally solved structure of the catalytic domain of I-TevI homing nuclease used as a modeling template. The predicted secondary structure of R.Eco29kI is shown above the alignment, the structure of I-TevI is shown below the alignment (H, helix; e, extended). Identical residues are shown in black, the other conserved residues are in grey. Amino acid residues analyzed in this work are labeled.

Figure 2

Comparison of the modeled structure of R.Eco29kI (a) and the catalytic domain of I-TevI (b). Secondary structures are colored (helices in red, strands in orange). Amino acid residues of the catalytic pocket in both enzymes and the non-essential R86 residue of R.Eco29kI are indicated and labeled. Positively charged residues are shown in blue, negatively charged residues are shown in red, neutral polar residues are shown in yellow.

Figure 3

DNA binding specificity of wt R.Eco29kI and mutant proteins. (a) Effect of Mg²⁺ ions on DNA binding by wt R.Eco29kI and R86A mutant. Lane 1, pUC128 PstI-PvuII fragments; lanes 2 and 3, reactions were carried out in the presence of Mg²⁺ ions; lanes 4 and 5, without Mg²⁺ ions. (b) Gel shift analysis of DNA binding by the mutant proteins in the presence of 5 mM EDTA. The names of the lanes are shown above the gel.

Figure 4

Binding of wt R.Eco29kI (a), H108F (b) and N154L (c) mutants to 24-mer DNA duplex containing the CCGCGG R.Eco29kI recognition site. The K_d values were determined by direct titration using the 24-mer DNA duplex as the fluorescent probe. Serial dilutions of wild type and mutant proteins were incubated with 1 nM FAM-labeled duplex.

Figure 5

DNA cleavage activity of wt R.Eco29kI and its mutants. (a) Electrophoresis of the reaction products of phage φ80vir DNA with R.Eco29kI and mutant proteins in 0.8% agarose. (b) Electrophoresis of the cleavage products of the 200 bp DNA fragment by R.Eco29kI and mutant proteins in 5 % polyacrylamide gel under non-denaturing conditions. (c) Electrophoresis of the reaction products of the 200 bp DNA fragment with R.Eco29kI and mutant proteins in 8% polyacrylamide gel under denaturing conditions. Gel patterns with cleavage products are shown at the points after which the reaction rate ceased to rise. s – substrate, 200 bp DNA fragment with Eco29kI site; p – products, the cleavage products of 200 bp DNA fragment after treatment by R.Eco29kI.