title: Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family.
abstract: Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.
authors: Graduate Program in Molecular and Cellular Biology and the Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98019, USA.
authors: Gregory K
authors: GK
authors: Taylor
authors: Daniel F
authors: DF
authors: Heiter
authors: Shmuel
authors: S
authors: Pietrokovski
authors: Barry L
authors: BL
authors: Stoddard
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D008958:Models, Molecular; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C071421:vsr endonuclease