title: To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
abstract: Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
authors.forename: Samantha S
authors.initials: SS
authors.lastname: Katz
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Frederick S
authors.initials: FS
authors.lastname: Gimble
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
authors.forename: Francesca
authors.initials: F
authors.lastname: Storici
mesh_terms: D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D053837:DNA Cleavage; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D009154:Mutation; D051135:Rad51 Recombinase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics*
chemical_list: D051135:Rad51 Recombinase; D003850:Deoxyribonuclease I