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To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

Katz SS, Gimble FS, Storici F - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

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.

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DNA cleavage activities of the wild-type, D145A, and K223I I-SceI proteins.(A) Scheme of the 18-bp I-SceI recognition sequence showing the cleavage positions of wild-type I-SceI and K223I I-SceI. Supercoiled pBS-I-SceI (E/H) plasmid DNA was incubated with (B) wild-type I-SceI, (C) D145A I-SceI mutant, or (D) K223I I-SceI mutant for various lengths of time and the amounts of the nicked open circle (orange circles) and linear (blue squares) reaction product DNAs were plotted as a function of time. Data points represent the average values of two experiments. Insets show the same data immediately following initiation of the reactions.
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pone-0088840-g001: DNA cleavage activities of the wild-type, D145A, and K223I I-SceI proteins.(A) Scheme of the 18-bp I-SceI recognition sequence showing the cleavage positions of wild-type I-SceI and K223I I-SceI. Supercoiled pBS-I-SceI (E/H) plasmid DNA was incubated with (B) wild-type I-SceI, (C) D145A I-SceI mutant, or (D) K223I I-SceI mutant for various lengths of time and the amounts of the nicked open circle (orange circles) and linear (blue squares) reaction product DNAs were plotted as a function of time. Data points represent the average values of two experiments. Insets show the same data immediately following initiation of the reactions.

Mentions: To determine the efficacy and the mechanism of SSB-driven gene targeting, we chose a nickase whose activity could be directly compared to variant forms of the same enzyme that do not cleave DNA or that cleave both DNA strands. These different variants of the double-strand endonuclease I-SceI homing enzyme were generated previously [31]. K223I I-SceI contains an amino acid substitution that borders the catalytic center and rapidly cleaves one specific DNA strand at the cognate 18-bp I-SceI recognition sequence but not the other (Figure 1A) without producing a hairpin product [31]. D145A I-SceI is a variant enzyme that contains an alanine substitution of an essential active site residue and is completely defective in DNA cleavage activity [31]. To compare the in vitro cleavage activities of the wild-type, K223I, and D145A I-SceI proteins, a supercoiled plasmid (pBS-I-SceI (E/H)) containing a single I-SceI recognition sequence was incubated with each of these proteins. Within 20 minutes, both DNA strands of the plasmid were completely cleaved by the wild-type I-SceI protein to yield the linearized product (Figure 1B), but the D145A I-SceI variant failed to cleave the plasmid DNA even after 12 hours (Figure 1C). The K223I I-SceI variant produced a nicked open circular product within 20 minutes which was slowly converted into the linearized form over many hours (Figure 1D), indicating that the enzyme efficiently generates an SSB but not a DSB. K223I nicking rate is approximately the same as the double-strand cleavage rate by wild-type I-SceI. We estimate that nicking of the DNA by the K223I I-SceI variant occurs approximately 180-fold faster than linearization.


To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

Katz SS, Gimble FS, Storici F - PLoS ONE (2014)

DNA cleavage activities of the wild-type, D145A, and K223I I-SceI proteins.(A) Scheme of the 18-bp I-SceI recognition sequence showing the cleavage positions of wild-type I-SceI and K223I I-SceI. Supercoiled pBS-I-SceI (E/H) plasmid DNA was incubated with (B) wild-type I-SceI, (C) D145A I-SceI mutant, or (D) K223I I-SceI mutant for various lengths of time and the amounts of the nicked open circle (orange circles) and linear (blue squares) reaction product DNAs were plotted as a function of time. Data points represent the average values of two experiments. Insets show the same data immediately following initiation of the reactions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928301&req=5

pone-0088840-g001: DNA cleavage activities of the wild-type, D145A, and K223I I-SceI proteins.(A) Scheme of the 18-bp I-SceI recognition sequence showing the cleavage positions of wild-type I-SceI and K223I I-SceI. Supercoiled pBS-I-SceI (E/H) plasmid DNA was incubated with (B) wild-type I-SceI, (C) D145A I-SceI mutant, or (D) K223I I-SceI mutant for various lengths of time and the amounts of the nicked open circle (orange circles) and linear (blue squares) reaction product DNAs were plotted as a function of time. Data points represent the average values of two experiments. Insets show the same data immediately following initiation of the reactions.
Mentions: To determine the efficacy and the mechanism of SSB-driven gene targeting, we chose a nickase whose activity could be directly compared to variant forms of the same enzyme that do not cleave DNA or that cleave both DNA strands. These different variants of the double-strand endonuclease I-SceI homing enzyme were generated previously [31]. K223I I-SceI contains an amino acid substitution that borders the catalytic center and rapidly cleaves one specific DNA strand at the cognate 18-bp I-SceI recognition sequence but not the other (Figure 1A) without producing a hairpin product [31]. D145A I-SceI is a variant enzyme that contains an alanine substitution of an essential active site residue and is completely defective in DNA cleavage activity [31]. To compare the in vitro cleavage activities of the wild-type, K223I, and D145A I-SceI proteins, a supercoiled plasmid (pBS-I-SceI (E/H)) containing a single I-SceI recognition sequence was incubated with each of these proteins. Within 20 minutes, both DNA strands of the plasmid were completely cleaved by the wild-type I-SceI protein to yield the linearized product (Figure 1B), but the D145A I-SceI variant failed to cleave the plasmid DNA even after 12 hours (Figure 1C). The K223I I-SceI variant produced a nicked open circular product within 20 minutes which was slowly converted into the linearized form over many hours (Figure 1D), indicating that the enzyme efficiently generates an SSB but not a DSB. K223I nicking rate is approximately the same as the double-strand cleavage rate by wild-type I-SceI. We estimate that nicking of the DNA by the K223I I-SceI variant occurs approximately 180-fold faster than linearization.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

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.

Show MeSH