title: 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.
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
authors.forename: Iwona A
authors.initials: IA
authors.lastname: Cymerman
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Obarska
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Skowronek
authors.forename: Arvydas
authors.initials: A
authors.lastname: Lubys
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Bujnicki
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011487:Protein Conformation; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; C024412:GGTGA-specific type II deoxyribonucleases