Mutations altering the cleavage specificity of a homing endonuclease

Lenny M. Seligman; Karen M. Chisholm; Brett S. Chevalier; Meggen S. Chadsey; Samuel T. Edwards; Jeremiah H. Savage; Adeline L. Veillet

doi:10.1093/nar/gkf495

Mutations altering the cleavage specificity of a homing endonuclease

Lenny M. Seligman
, Karen M. Chisholm
, Brett S. Chevalier
, Meggen S. Chadsey
, Samuel T. Edwards
, Jeremiah H. Savage
, Adeline L. Veillet

Research output: Contribution to journal › Article › peer-review

106 Scopus citations

Abstract

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.

Original language	English (US)
Pages (from-to)	3870-3879
Number of pages	10
Journal	Nucleic acids research
Volume	30
Issue number	17
DOIs	https://doi.org/10.1093/nar/gkf495
State	Published - Sep 1 2002
Externally published	Yes

Funding

We thank Alexis Kaushansky, Cheryl Okumura and Saul Rosenstein for experimental contributions and Colin Manoil for valuable comments on the manuscript. Special thanks are due to Ray Monnat and Barry Stoddard for numerous contributions, both material and intellectual. This work was supported by a grant to L.M.S. from the NSF (RUI-9870817). K.M.C. was also supported by a grant to Pomona College from the Howard Hughes Medical Institute. M.S.C. and B.S.C. were supported by NCI grant CA88942 and by a Molecular Training Grant in Cancer Research (T32 CA 09437, to M.S.C.) and an Interdisciplinary Training Grant in Cancer Research (T32 CA80416, to B.S.C).

Funders	Funder number
Author National Science Foundation National Science Foundation National Institutes of Health National Institutes of Health National Institutes of Health National Institutes of Health National Science Foundation National Science Foundation	RUI-9870817
Howard Hughes Medical Institute
National Institute of Health-National Cancer Institute	T32CA080416, CA88942, T32 CA 09437

ASJC Scopus subject areas

Genetics

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10.1093/nar/gkf495

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title = "Mutations altering the cleavage specificity of a homing endonuclease",

abstract = "The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.",

author = "Seligman, \{Lenny M.\} and Chisholm, \{Karen M.\} and Chevalier, \{Brett S.\} and Chadsey, \{Meggen S.\} and Edwards, \{Samuel T.\} and Savage, \{Jeremiah H.\} and Veillet, \{Adeline L.\}",

note = "Funding Information: We thank Alexis Kaushansky, Cheryl Okumura and Saul Rosenstein for experimental contributions and Colin Manoil for valuable comments on the manuscript. Special thanks are due to Ray Monnat and Barry Stoddard for numerous contributions, both material and intellectual. This work was supported by a grant to L.M.S. from the NSF (RUI-9870817). K.M.C. was also supported by a grant to Pomona College from the Howard Hughes Medical Institute. M.S.C. and B.S.C. were supported by NCI grant CA88942 and by a Molecular Training Grant in Cancer Research (T32 CA 09437, to M.S.C.) and an Interdisciplinary Training Grant in Cancer Research (T32 CA80416, to B.S.C).",

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AU - Edwards, Samuel T.

AU - Savage, Jeremiah H.

AU - Veillet, Adeline L.

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Mutations altering the cleavage specificity of a homing endonuclease

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