I-CreI

I-"Cre"I is a homing endonuclease whose gene was first discovered in the chloroplast genome of "Chlamydomonas reinhardtii", a species of unicellular green algae.Rochaix JD, Malnoe P. (1978) Anatomy of the chloroplast ribosomal DNA of "Chlamydomonas reinhardtii". "Cell". 15:661-670. ] It is named for the facts that: it resides in an Intron; it was isolated from "Clamydomonas reinhardtii"; it was the first (I) such gene isolated from "C. reinhardtii". Interestingly, its gene resides in a group I intron in the 23S ribosomal RNA gene of the "C. reinhardtii" chloroplast, and I-"Cre"I is only expressed when its mRNA is spliced from the primary transcript of the 23S gene. I-"Cre"I enzyme, which functions as a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-"Cre"I is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-"Cre"I-containing intron encounters a 23S gene lacking the intron, I-"Cre"I enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-"Cre"I provides for its own propagation while conferring no benefit on its host, it is an example of selfish DNA.

Discovery

I-"Cre"I was first observed as an intervening sequence in the 23S rRNA gene of the "C. reinhardtii" chloroplast genome. The 23S gene is an RNA gene, meaning that its transcript is not translated into protein. As RNA, it forms part of the large subunit of the ribosome. An open reading frame coding for a 163-amino acid protein was found in this 23S intron, suggesting that a protein might facilitate the homing behavior of the mobile intron. Furthermore, the predicted protein had a LAGLIDADG motif, a conserved amino acid sequence that is present in other proteins coded for in group I mobile introns. A 1991 study established that the ORF codes for a DNA endonuclease, I-"Cre"I, which selectively cuts a site corresponding to where the intron is spliced out of the 23S primary transcript.Dürrenberger F, Rochaix JD. (1991) Chloroplast ribosomal intron of "Chlamydomonas reinhardtii": "in vitro" self-splicing, DNA endonuclease activity and "in vivo" mobility. "EMBO J". 10:3495-3501 ] The study also showed that the intron was able to invade 23S alleles that did not already have it.

Mechanism of propagation

I-"Cre"I has evolved to cut a 22-nucleotide sequence of DNA that occurs in alleles of the 23S ribosomal RNA gene that lack the I-"Cre"I-containing intron. When such an "intron-minus" allele is cut, pathways of double-strand break repair are activated in the cell. The cell uses as a template for repair the 23S allele that yielded the responsible I-"Cre"I enzyme, thus replicating the I-"Cre"I-containing intron.Dürrenberger F, Thompson AJ, Herrin DL, Rochaix JD. (1996) Double strand break-induced recombination in "Chlamydomonas reinhardtii" chloroplasts. "Nucleic Acids Research". 24:3323-3331. ] The resulting "intron-plus" allele no longer contains an intact homing site for the I-"Cre"I enzyme, and is therefore not cleaved. Since this intron provides for its own replication without conferring any benefit on its host, I-"Cre"I is a form of selfish DNA.

tructural studies and possible applications

outside of the gene of interest.

In order to use I-"Cre"I as a tool in this fashion, it is necessary to make it recognize and cleave sequences of DNA different from its native homing site. An "Escherichia coli" genetic system for studying the relationship between I-"Cre"I structure and its homing site specificity was created in 1997.Seligman LM, Stephens KM, Savage JH, Monnat RJ. (1997) Genetic Analysis of the "Chlamydomonas reinhardtii" I-"Cre"I Mobile intron Homing System in "Escherichia coli". "Genetics". 147:1653-1664. ] In 1998, the crystal structure of I-"Cre"I enzyme bound to its native homing site was solved, greatly aiding research in altering the homing site recognition of the protein. Mutant forms of the protein have since been created that exhibit altered homing site specificity.Seligman LM, Chisholm KM, Chevalier BS, Chadsey MS, Edwards ST, Savage JH, Veillet AL. (2002) Mutations altering the cleavage specificity of a homing endonuclease. "Nucleic Acids Research". 30:3870-3879. ] Sussman D, Chadsey M, Fauce S, Engel A, Bruett A, Monnat R, Stoddard BL, Seligman LM. (2004) Isolation and Characterization of New Homing Endonuclease Specificities at Individual Target Site Positions. "Journal of Molecular Biology". 342:31-41. ] Rosen LE, Morrison HA, Masri S, Brown MJ, Springstubb B, Sussman D, Stoddard BL, Seligman LM. (2006) Homing endonuclease I-"Cre"I derivatives with novel DNA target specificities. "Nucleic Acids Research". 34:4791-4800. ] A genetic system in "Saccharomyces cerevisiae" has also been created, yielding additional I-"Cre"I mutants with modified homing site specificities.Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat JC, Stricher F, Petit AS, Patin A, Guillier S, Rolland S, Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, Duchateau P, Pâques F. (2006) Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets. "Journal of Molecular Biology". 355:443-458. ] Smith J, Grizot S, Arnould S, Duclert A, Epinat JC, Chames P, Prieto J, Redondo P, Blanco FJ, Bravo J, Montoya G, Pâques F, Duchateau P. (2006) A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. "Nucleic Acids Research". In press. ]

I-"Cre"I has already been used successfully to induce homologous recombination in "Drosophila melanogaster", an extremely popular eukaryotic model organism.Maggert KA, Golic KG. (2005) Highly Efficient Sex Chromosome Interchanges Produced By I-"Cre"I Expression in "Drosophila". "Genetics". 171:1103-1114. ] It seems very likely that advances in molecular biological techniques and generation of a library of I-"Cre"I-derived novel endonucleases will eventually allow for the targeting of many genes of etiological significance.

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