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PMID: 7905633 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Chimeric restriction endonuclease.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 91 ·No. 3 ·1994-02-01 ·Pages 883-7

Kim YG, Chandrasegaran S

Abstract

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nt away from the recognition site. Recently, we reported the presence of two distinct and separable domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA-binding domain) and the other for the endonuclease activity (the cleavage domain). Here, we report the construction of a chimeric restriction endonuclease by linking the Drosophila Ultrabithorax homeodomain to the cleavage domain (FN) of Fok I restriction endonuclease. The hybrid enzyme, Ubx-FN, was purified, and its cleavage properties were characterized. The hybrid enzyme shows the same DNA sequence-binding preference as that of Ubx; as expected, it cleaves the DNA away from the recognition site. On the 5'-TTAATGGTT-3' strand the hybrid enzyme cleaves 3 nt away from the recognition site, whereas it cuts the complementary 5'-AACCATTAA-3' strand 8, 9, or 10 nt away from the binding site. Similarly engineered hybrid enzymes could be valuable tools in physical mapping and sequencing of large eukaryotic genomes.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Binding Sites/genetics Cloning, Molecular DNA, Recombinant/chemistry,genetics Deoxyribonucleases, Type II Site-Specific/genetics Drosophila/genetics Escherichia coli/genetics Flavobacterium/enzymology,genetics Genes, Bacterial Genes, Homeobox Genes, Insect Genetic Vectors Molecular Sequence Data Nucleic Acid Conformation Protein Conformation Recombinant Fusion Proteins/chemistry,genetics
Chemicals
DNA, Recombinant Recombinant Fusion Proteins endodeoxyribonuclease FokI Deoxyribonucleases, Type II Site-Specific
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Kim Y G
Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205-2179.
Chandrasegaran S
References (26)
26 references, click to expand
  1. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  2. C-terminal deletion mutants of the FokI restriction endonuclease.
    Gene. 1993 Oct 29;133(1):79-84 PMID: 8224897
  3. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  4. New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI).
    Gene. 1981 Dec;16(1-3):73-8 PMID: 6282705
  5. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues.
    J Chromatogr. 1987 Dec 18;411:177-84 PMID: 3443622
  6. Conferring operator specificity on restriction endonucleases.
    Science. 1988 Aug 26;241(4869):1084-6 PMID: 2842862
  7. The fokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes.
    J Biol Chem. 1989 Apr 5;264(10):5751-6 PMID: 2784436
  8. The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors.
    Cell. 1989 Nov 3;59(3):573-80 PMID: 2572329
  9. Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase.
    Gene. 1989 Aug 15;80(2):193-208 PMID: 2684765
  10. Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
    Proc Natl Acad Sci U S A. 1990 Apr;87(8):2882-6 PMID: 2158096
  11. Construction of an efficient overproducer clone of HinfI restriction endonuclease using the polymerase chain reaction.
    Gene. 1990 Mar 30;88(1):1-5 PMID: 2187744
  12. Use of T7 RNA polymerase to direct expression of cloned genes.
    Methods Enzymol. 1990;185:60-89 PMID: 2199796
  13. Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions.
    Cell. 1990 Nov 2;63(3):579-90 PMID: 1977522
  14. What determines the specificity of action of Drosophila homeodomain proteins?
    Cell. 1990 Nov 30;63(5):883-94 PMID: 1979524
  15. Optimal DNA sequence recognition by the Ultrabithorax homeodomain of Drosophila.
    EMBO J. 1991 May;10(5):1179-86 PMID: 1673656
  16. Class-IIS restriction enzymes--a review.
    Gene. 1991 Apr;100:13-26 PMID: 2055464
  17. Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.
    Nucleic Acids Res. 1991 Oct 11;19(19):5233-6 PMID: 1923807
  18. Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.
    Cell. 1991 Nov 1;67(3):517-28 PMID: 1682054
  19. Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage.
    Science. 1991 Dec 6;254(5037):1494-7 PMID: 1962209
  20. Site-specific cleavage of human chromosome 4 mediated by triple-helix formation.
    Science. 1991 Dec 13;254(5038):1639-42 PMID: 1836279
  21. A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC).
    Gene. 1992 Jan 2;110(1):1-7 PMID: 1531969
  22. Functional domains in Fok I restriction endonuclease.
    Proc Natl Acad Sci U S A. 1992 May 15;89(10):4275-9 PMID: 1584761
  23. Differential DNA sequence recognition is a determinant of specificity in homeotic gene action.
    EMBO J. 1992 Nov;11(11):4059-72 PMID: 1356765
  24. Conferring new cleavage specificities of restriction endonucleases.
    Methods Enzymol. 1992;216:321-9 PMID: 1336096
  25. Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis.
    Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2764-8 PMID: 8464886
  26. A new method for sequencing DNA.
    Proc Natl Acad Sci U S A. 1977 Feb;74(2):560-4 PMID: 265521
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.91.3.883
Published
1994-02-01
Pages
883-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC521416
Subset
IM
Grants
NIGMS NIH HHS · GM 42140 · United States
External Links
PubMed source DOI: 10.1073/pnas.91.3.883 PMC Full Text View journal details
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