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The mechanism of gene targeting in human somatic cells.

Kan Y, Ruis B, Lin S, Hendrickson EA - PLoS Genet. (2014)

Bottom Line: Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector.Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process.These observations significantly advance our understanding of HR and gene targeting in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

ABSTRACT
Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

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Related in: MedlinePlus

rAAV gene targeting efficiency correlates with HR, and not single strand annealing, activity.(A) The HR assay. SceGFP is a full-length GFP gene disrupted by an I-SceI site. HR between SceGFP and the internal GFP (iGFP) fragment on the same plasmid upon I-SceI digestion restores GFP activity. (B) The single strand annealing assay. 5′GFP and 3′SceGFP are GFP fragments bearing 266 bp of homology. Single strand annealing repair of the I-SceI-induced DSB generates a functional GFP gene. (C) The efficiency of HR, single strand annealing and rAAV gene targeting. The indicated cell lines were analyzed using the HR and single strand annealing assays as well as for rAAV gene targeting. The mean ± SEM of three independent experiments is shown.
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pgen-1004251-g004: rAAV gene targeting efficiency correlates with HR, and not single strand annealing, activity.(A) The HR assay. SceGFP is a full-length GFP gene disrupted by an I-SceI site. HR between SceGFP and the internal GFP (iGFP) fragment on the same plasmid upon I-SceI digestion restores GFP activity. (B) The single strand annealing assay. 5′GFP and 3′SceGFP are GFP fragments bearing 266 bp of homology. Single strand annealing repair of the I-SceI-induced DSB generates a functional GFP gene. (C) The efficiency of HR, single strand annealing and rAAV gene targeting. The indicated cell lines were analyzed using the HR and single strand annealing assays as well as for rAAV gene targeting. The mean ± SEM of three independent experiments is shown.

Mentions: To confirm that rAAV gene targeting efficiency correlated with HR, and not single strand assimilation, activity, we transfected HCT116 cells with Rad51K133A, a dominant negative form of Rad51 reported to reduce HR and concomitantly elevate single strand annealing [23]. Using episomal reporters for either HR (Figure 4A) or single strand annealing (Figure 4B), we confirmed that expression of the dominant negative indeed reduced HR and increased single strand annealing in HCT116 cells (Figure 4C). Importantly, the rAAV targeting efficiency at the HPRT locus was reduced by 6.2-fold upon Rad51K133A transfection, which correlated well with the reduced HR activity and not the increased single strand annealing activity in these cells (Figure 4C). Thus, consistent with the sectoring assay, this result further confirmed that rAAV gene targeting is mediated predominantly by HR instead of single strand assimilation in human cells.


The mechanism of gene targeting in human somatic cells.

Kan Y, Ruis B, Lin S, Hendrickson EA - PLoS Genet. (2014)

rAAV gene targeting efficiency correlates with HR, and not single strand annealing, activity.(A) The HR assay. SceGFP is a full-length GFP gene disrupted by an I-SceI site. HR between SceGFP and the internal GFP (iGFP) fragment on the same plasmid upon I-SceI digestion restores GFP activity. (B) The single strand annealing assay. 5′GFP and 3′SceGFP are GFP fragments bearing 266 bp of homology. Single strand annealing repair of the I-SceI-induced DSB generates a functional GFP gene. (C) The efficiency of HR, single strand annealing and rAAV gene targeting. The indicated cell lines were analyzed using the HR and single strand annealing assays as well as for rAAV gene targeting. The mean ± SEM of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974634&req=5

pgen-1004251-g004: rAAV gene targeting efficiency correlates with HR, and not single strand annealing, activity.(A) The HR assay. SceGFP is a full-length GFP gene disrupted by an I-SceI site. HR between SceGFP and the internal GFP (iGFP) fragment on the same plasmid upon I-SceI digestion restores GFP activity. (B) The single strand annealing assay. 5′GFP and 3′SceGFP are GFP fragments bearing 266 bp of homology. Single strand annealing repair of the I-SceI-induced DSB generates a functional GFP gene. (C) The efficiency of HR, single strand annealing and rAAV gene targeting. The indicated cell lines were analyzed using the HR and single strand annealing assays as well as for rAAV gene targeting. The mean ± SEM of three independent experiments is shown.
Mentions: To confirm that rAAV gene targeting efficiency correlated with HR, and not single strand assimilation, activity, we transfected HCT116 cells with Rad51K133A, a dominant negative form of Rad51 reported to reduce HR and concomitantly elevate single strand annealing [23]. Using episomal reporters for either HR (Figure 4A) or single strand annealing (Figure 4B), we confirmed that expression of the dominant negative indeed reduced HR and increased single strand annealing in HCT116 cells (Figure 4C). Importantly, the rAAV targeting efficiency at the HPRT locus was reduced by 6.2-fold upon Rad51K133A transfection, which correlated well with the reduced HR activity and not the increased single strand annealing activity in these cells (Figure 4C). Thus, consistent with the sectoring assay, this result further confirmed that rAAV gene targeting is mediated predominantly by HR instead of single strand assimilation in human cells.

Bottom Line: Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector.Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process.These observations significantly advance our understanding of HR and gene targeting in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

ABSTRACT
Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

Show MeSH
Related in: MedlinePlus