Premium
Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease
Author(s) -
Cymerman Iwona A.,
Obarska Agnieszka,
Skowronek Krzysztof J.,
Lubys Arvydas,
Bujnicki Janusz M.
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21156
Subject(s) - genetics , subfamily , restriction enzyme , biology , dna , nuclease , endonuclease , context (archaeology) , computational biology , gene , paleontology
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. Proteins 2006. © 2006 Wiley‐Liss, Inc.