Identification of a Single HNH Active Site in Type IIS Restriction Endonuclease Eco31I

Abstract

SUMMARYType IIS restriction endonuclease Eco31I is a 'short-distance cutter', which cleaves DNA strands close to its recognition sequence, 5′-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in Type IIS restriction endonucleases that recognize common sequence core GTCTC.

Evolution and classification of the CRISPR–Cas systems

“…Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein, but the function of these domains remains to be elucidated. However, as the HNH nuclease domain is abundant in restriction enzymes and possesses endonuclease activity 37,38 , it is likely to be responsible for target cleavage. Furthermore, for the S. thermophilus type II CRISPR–Cas system, targeting of plasmid and phage DNA has been demonstrated in vivo 20 and inactivation of Cas9 has been shown to abolish interference 16 .…”

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

“…4b). 19,20 PD-(D/E) XK superfamily restriction enzymes SfiI, Cfr10I, Bse634I, and NgoMIV, 11,[21][22][23] similarly to Cfr42I, function as homotetramers (Fig. 4c).…”

The role of the N-terminal loop in the function of the colicin E7 nuclease domain

“…I-Hmu-I [18]), procaryotic extracellular nucleases (Nuclease A [19]) and also in increasing number of restriction endonucleases (e.g. MnlI [20], KpnI [21,22], HphI [23], Eco31I [24], Hpy99I [25]). Sequences are collected in the HNH [26] and Pfam databases [27].…”