Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

Abstract

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.

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].…”

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

“…27 The HNH active site was also predicted by bioinformatics 28 and later demonstrated experimentally to be present in various Type II REases, including KpnI, 29 MnlI, 7 and HphI. 10 Structurally, the ββα-Me finger forms an antiparallel β-hairpin followed by an α-helix, which provide a scaffold for binding of a metal ion by residues located in the first strand and in the helix. The above-mentioned enzymes have different overall three-dimensional structures, suggesting that the common catalytic module has been inserted into evolutionarily unrelated domains or independently developed different structural elaborations that stabilize the minimal ββα core.…”

“…9 Bioinformatic and mutational analysis of Type IIS REase HphI have also provided evidence that this enzyme has a catalytic domain related to the HNH nucleases. 10 The HNH motif was also indicated in KpnI, which belongs to Type IIP REases, the single domain enzymes. The analysis of KpnI mutants showed that HNH motif of KpnI is coupled with DNA recognition function, contrary to the HNH motifs indicated in Type IIS REases.…”

Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif

“…11-13). HNH proteins include a range of nucleases such as some homing endonucleases (e.g., I-HmuI), colicins (e.g., ColE7, ColE9), and restriction endonucleases (e.g., KpnI, MnlI, and HphI) (14)(15)(16)(17)(18)(19). HNH motifs are less common in eukaryotes.…”