Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006;34(17):4791-800.
doi: 10.1093/nar/gkl645. Epub 2006 Sep 13.

Homing endonuclease I-CreI derivatives with novel DNA target specificities

Laura E Rosen  1 , Holly A Morrison, Selma Masri, Michael J Brown, Brendan Springstubb, Django Sussman, Barry L Stoddard, Lenny M Seligman
Affiliations

Homing endonuclease I-CreI derivatives with novel DNA target specificities

Laura E Rosen et al. Nucleic Acids Res. 2006.

Abstract

Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins.

PubMed Disclaimer

Figures

Figure 1
Figure 1
I-CreI/DNA contacts. Direct protein/DNA hydrogen bonds are red; water molecules and water-mediated hydrogen bonds are blue. Palindromic bases are green; non-palindromic are white. Scissile phosphate groups are indicated with black dots.
Figure 2
Figure 2
In vivo endonuclease assays. Colony phenotypes resulting from transforming indicated I-CreI derivatives into cells harboring the indicated target site mutants (13). Increased proportion of white cells within colonies indicates increased site affinity. The Q26C/Y66R mutant has been described previously (14).
Figure 3
Figure 3
Relative cleavage of cognate and non-cognate targets. Wild-type (left) and Q44V (right) endonucleases were exposed to linearized plasmids containing wild-type and A:T ±4 homing sites. The left lanes in each gel (upper) indicate the enzyme concentration required for complete digestion of each substrate. Serial 2-fold dilutions were performed, and the relative enzyme concentrations required for 50% cleavage of each substrate determined (lower).
Figure 4
Figure 4
Structures and interactions of cognate pairs at residue 44 and base pair ±4 and residue 28 and base pair ±7. Protein/DNA contacts between wild-type I-CreI and its binding site are shown in (A and C). Contacts between Q44V and K28R and their cognate sites are shown in (B and D), respectively. The sequences of wild-type I-CreI target sequence and the alternate target sequences are shown below their corresponding structures. Dashed green lines represent hydrogen bonding and dashed magenta lines represent van der Waals interactions.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Belfort M., Roberts R.J. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 1997;25:3379–3388. - PMC - PubMed
    1. Chevalier B.S., Stoddard B.L. Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 2001;29:3757–3774. - PMC - PubMed
    1. Gimble F.S. Invasion of a multitude of genetic niches by mobile endonuclease genes. FEMS Microbiol. Lett. 2000;185:99–107. - PubMed
    1. Burt A., Koufopanou V. Homing endonuclease genes: the rise and fall and rise again of a selfish element. Curr. Opin. Genet. Dev. 2004;14:609–615. - PubMed
    1. Seligman L.M., Stephens K.M., Savage J.H., Monnat R.J., Jr Genetic analysis of the Chlamydomonas reinhardtii I-CreI mobile intron homing system in Escherichia coli. Genetics. 1997;147:1653–1664. - PMC - PubMed

Publication types