Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI

Kenji Ichiyanagi, Yoshizumi Ishino, Mariko Ariyoshi, Kayoko Komori, Kosuke Morikawa

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 Å crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded β-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)889-901
Number of pages13
JournalJournal of Molecular Biology
Volume300
Issue number4
DOIs
Publication statusPublished - Jul 21 2000
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

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