Chimeric restriction enzymes are a novel class of engineered nucleases in which the nonspecific DNA cleavage domain (a type IIs restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely, the zinc finger motif, the helix-turn-helix motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by micro-injection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of DNA into chromatin. In addition, this cleavage activated the target modules for efficient homologous recombination. The goal here is to redesign and improve the chimeric nucleases by computer modeling based on the crystal structures of native FokI and FokI bound to DNA. The investigators also plan to increase the sequence specificity of the hybrid endonucleases by using polydactyl zinc fingers. They plan to induce homologous recombination at a chromosomal site by using engineered chimeric nucleases within a human or animal cell line. Their long term goal is the application of chimeric nucleases to correct a genetic defect in an animal model.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053923-07
Application #
6519711
Study Section
Genome Study Section (GNM)
Program Officer
Lewis, Catherine D
Project Start
1996-05-01
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
7
Fiscal Year
2002
Total Cost
$276,750
Indirect Cost
Name
Johns Hopkins University
Department
Public Health & Prev Medicine
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Chandrasegaran, Srinivasan; Carroll, Dana (2016) Origins of Programmable Nucleases for Genome Engineering. J Mol Biol 428:963-89
Mani, Mala; Kandavelou, Karthikeyan; Dy, Fei Jamie et al. (2005) Design, engineering, and characterization of zinc finger nucleases. Biochem Biophys Res Commun 335:447-57
Mani, Mala; Smith, Jeff; Kandavelou, Karthikeyan et al. (2005) Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage. Biochem Biophys Res Commun 334:1191-7
Choe, Wonchae; Chandrasegaran, Srinivasan; Ostermeier, Marc (2005) Protein fragment complementation in M.HhaI DNA methyltransferase. Biochem Biophys Res Commun 334:1233-40
Durai, Sundar; Mani, Mala; Kandavelou, Karthikeyan et al. (2005) Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33:5978-90
Bibikova, M; Carroll, D; Segal, D J et al. (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21:289-97
Ruminy, P; Derambure, C; Chandrasegaran, S et al. (2001) Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease. J Mol Biol 310:523-35
Smith, J; Bibikova, M; Whitby, F G et al. (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28:3361-9
Smith, J; Berg, J M; Chandrasegaran, S (1999) A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acids Res 27:674-81
Chandrasegaran, S; Smith, J (1999) Chimeric restriction enzymes: what is next? Biol Chem 380:841-8

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