Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold

TY - JOUR

T1 - Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold

AU - Molina, Rafael

AU - Marcaida, María José

AU - Redondo, Pilar

AU - Marenchino, Marco

AU - Duchateau, Phillippe

AU - D'Abramo, Marco

AU - Montoya, Guillermo

AU - Prieto, Jesús

N1 - © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PY - 2015/7/24

Y1 - 2015/7/24

N2 - Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous recombination, an error-free mechanism, or by non-homologous end joining, a process susceptible to introducing errors in the repaired sequence. The type of DNA cleavage might alter the balance between these two alternatives. The use of "nickases" producing a specific single strand break instead of a double strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional.

AB - Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous recombination, an error-free mechanism, or by non-homologous end joining, a process susceptible to introducing errors in the repaired sequence. The type of DNA cleavage might alter the balance between these two alternatives. The use of "nickases" producing a specific single strand break instead of a double strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional.

KW - Amino Acid Motifs

KW - Catalytic Domain

KW - Circular Dichroism

KW - Crystallography, X-Ray

KW - DNA Breaks, Double-Stranded

KW - DNA End-Joining Repair

KW - DNA-Binding Proteins

KW - Deoxyribonuclease I

KW - Deoxyribonucleases, Type I Site-Specific

KW - Gene Targeting

KW - Homologous Recombination

KW - Molecular Dynamics Simulation

KW - Protein Engineering

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1074/jbc.m115.658666

DO - 10.1074/jbc.m115.658666

M3 - Journal article

C2 - 26045557

SN - 0021-9258

VL - 290

SP - 18534

EP - 18544

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 30

ER -