This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bibikova, M.
Right arrow Articles by Chandrasegaran, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bibikova, M.
Right arrow Articles by Chandrasegaran, S.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases

Marina Bibikova,1 Dana Carroll,1,* David J. Segal,1,dagger Jonathan K. Trautman,1 Jeff Smith,2,3 Yang-Gyun Kim,2,Dagger and Srinivasan Chandrasegaran2,*

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132,1 and Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health,2 and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine,3 Baltimore, Maryland 21205

Received 28 August 2000/Returned for modification 2 October 2000/Accepted 5 October 2000

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.

* Corresponding author. Mailing address for Dana Carroll: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132. Phone: (801) 581-5977. Fax: (801) 581-7959. E-mail: carroll{at}path.utah.edu. Mailing address for Srinivasan Chandrasegaran: Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 614-2289. Fax: (410) 955-0617. E-mail: chandra{at}welchlink.welch.jhu.edu.

dagger Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

Dagger Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • van Nierop, G. P., de Vries, A. A. F., Holkers, M., Vrijsen, K. R., Goncalves, M. A. F. V. (2009). Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease. Nucleic Acids Res 37: 5725-5736 [Abstract] [Full Text]  
  • Mittelman, D., Moye, C., Morton, J., Sykoudis, K., Lin, Y., Carroll, D., Wilson, J. H. (2009). Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells. Proc. Natl. Acad. Sci. USA 106: 9607-9612 [Abstract] [Full Text]  
  • Lippow, S. M., Aha, P. M., Parker, M. H., Blake, W. J., Baynes, B. M., Lipovsek, D. (2009). Creation of a type IIS restriction endonuclease with a long recognition sequence. Nucleic Acids Res 37: 3061-3073 [Abstract] [Full Text]  
  • Sanders, K. L., Catto, L. E., Bellamy, S. R. W., Halford, S. E. (2009). Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands. Nucleic Acids Res 37: 2105-2115 [Abstract] [Full Text]  
  • Beumer, K. J., Trautman, J. K., Bozas, A., Liu, J.-L., Rutter, J., Gall, J. G., Carroll, D. (2008). Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105: 19821-19826 [Abstract] [Full Text]  
  • Amacher, S. L. (2008). Emerging gene knockout technology in zebrafish: zinc-finger nucleases. Brief Funct Genomic Proteomic 7: 460-464 [Abstract] [Full Text]  
  • Chan, S.-h., Bao, Y., Ciszak, E., Laget, S., Xu, S.-y. (2007). Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities. Nucleic Acids Res 35: 6238-6248 [Abstract] [Full Text]  
  • Sander, J. D., Zaback, P., Joung, J. K., Voytas, D. F., Dobbs, D. (2007). Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35: W599-W605 [Abstract] [Full Text]  
  • Meng, X., Thibodeau-Beganny, S., Jiang, T., Joung, J. K., Wolfe, S. A. (2007). Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res 35: e81-e81 [Abstract] [Full Text]  
  • Horner, S. M., DiMaio, D. (2007). The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells. J. Virol. 81: 6254-6264 [Abstract] [Full Text]  
  • Moehle, E. A., Rock, J. M., Lee, Y.-L., Jouvenot, Y., DeKelver, R. C., Gregory, P. D., Urnov, F. D., Holmes, M. C. (2007). Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc. Natl. Acad. Sci. USA 104: 3055-3060 [Abstract] [Full Text]  
  • Morton, J., Davis, M. W., Jorgensen, E. M., Carroll, D. (2006). Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells. Proc. Natl. Acad. Sci. USA 103: 16370-16375 [Abstract] [Full Text]  
  • Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J. K., Carroll, D. (2006). Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases. Genetics 172: 2391-2403 [Abstract] [Full Text]  
  • Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M. H., Chandrasegaran, S. (2005). Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33: 5978-5990 [Abstract] [Full Text]  
  • Lloyd, A., Plaisier, C. L., Carroll, D., Drews, G. N. (2005). Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc. Natl. Acad. Sci. USA 102: 2232-2237 [Abstract] [Full Text]  
  • Doi, N., Kumadaki, S., Oishi, Y., Matsumura, N., Yanagawa, H. (2004). In vitro selection of restriction endonucleases by in vitro compartmentalization. Nucleic Acids Res 32: e95-e95 [Abstract] [Full Text]  
  • Carvin, C. D., Parr, R. D., Kladde, M. P. (2003). Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins. Nucleic Acids Res 31: 6493-6501 [Abstract] [Full Text]  
  • Uil, T. G., Haisma, H. J., Rots, M. G. (2003). Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. Nucleic Acids Res 31: 6064-6078 [Abstract] [Full Text]  
  • Hurt, J. A., Thibodeau, S. A., Hirsh, A. S., Pabo, C. O., Joung, J. K. (2003). Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc. Natl. Acad. Sci. USA 100: 12271-12276 [Abstract] [Full Text]  
  • Epinat, J.-C., Arnould, S., Chames, P., Rochaix, P., Desfontaines, D., Puzin, C., Patin, A., Zanghellini, A., Paques, F., Lacroix, E. (2003). A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res 31: 2952-2962 [Abstract] [Full Text]  
  • Miller, D. G., Petek, L. M., Russell, D. W. (2003). Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks. Mol. Cell. Biol. 23: 3550-3557 [Abstract] [Full Text]  
  • Porteus, M. H., Cathomen, T., Weitzman, M. D., Baltimore, D. (2003). Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks. Mol. Cell. Biol. 23: 3558-3565 [Abstract] [Full Text]  
  • Porteus, M. H., Baltimore, D. (2003). Chimeric Nucleases Stimulate Gene Targeting in Human Cells. Science 300: 763-763 [Full Text]  
  • Bibikova, M., Beumer, K., Trautman, J. K., Carroll, D. (2003). Enhancing Gene Targeting with Designed Zinc Finger Nucleases. Science 300: 764-764 [Full Text]  
  • Bibikova, M., Golic, M., Golic, K. G., Carroll, D. (2002). Targeted Chromosomal Cleavage and Mutagenesis in Drosophila Using Zinc-Finger Nucleases. Genetics 161: 1169-1175 [Abstract] [Full Text]  
  • Preston, C. R., Engels, W., Flores, C. (2002). Efficient Repair of DNA Breaks in Drosophila: Evidence for Single-Strand Annealing and Competition With Other Repair Pathways. Genetics 161: 711-720 [Abstract] [Full Text]  
  • Vasquez, K. M., Marburger, K., Intody, Z., Wilson, J. H. (2001). Manipulating the mammalian genome by homologous recombination. Proc. Natl. Acad. Sci. USA 98: 8403-8410 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»