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Nonspaced inverted DNA repeats are preferential targets for homology-directed gene repair in mammalian cells.

Holkers M, de Vries AA, Gonçalves MA - Nucleic Acids Res. (2011)

Bottom Line: Despite their patent biological relevance, the parameters governing DNA repeat-associated chromosomal transactions remain ill-defined.We found that nonspaced DNA repeats can, per se, engage the HR pathway of the cell and that this process is primarily dependent on their spacing and relative arrangement (i.e. parallel or antiparallel) rather than on their sequence.Indeed, our data demonstrate that contrary to direct and spaced inverted repeats, nonspaced inverted repeats are intrinsically recombinogenic motifs in mammalian cells lending experimental support to their role in genome dynamics in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.

ABSTRACT
DNA repeats constitute potential sites for the nucleation of secondary structures such as hairpins and cruciforms. Studies performed mostly in bacteria and yeast showed that these noncanonical DNA structures are breakage-prone, making them candidate targets for cellular DNA repair pathways. Possible culprits for fragility at repetitive DNA sequences include replication and transcription as well as the action of structure-specific nucleases. Despite their patent biological relevance, the parameters governing DNA repeat-associated chromosomal transactions remain ill-defined. Here, we established an episomal recombination system based on donor and acceptor complementary DNA templates to investigate the role of direct and inverted DNA repeats in homologous recombination (HR) in mammalian cells. This system allowed us also to ascertain in a stringent manner the impact of repetitive sequence replication on homology-directed gene repair. We found that nonspaced DNA repeats can, per se, engage the HR pathway of the cell and that this process is primarily dependent on their spacing and relative arrangement (i.e. parallel or antiparallel) rather than on their sequence. Indeed, our data demonstrate that contrary to direct and spaced inverted repeats, nonspaced inverted repeats are intrinsically recombinogenic motifs in mammalian cells lending experimental support to their role in genome dynamics in higher eukaryotes.

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Effect of repetitive DNA sequences in a direct or inverted repeat configuration on homology-directed gene repair in mammalian cells. (A) Flow cytometric analysis of human embryonic kidney 293T cells (HEK), human fetal retinoblasts (911 and PER.tTA.Cre76 [PER]) and African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorDR.1 or acceptorIR.1. Negative and positive controls for HR-mediated GFP repair in each of the tested cell types were provided by cells containing GFPΔATG and acceptorScR (−) or these two plasmids as well as pCAG.I-SceI (+), respectively. (B) Dot plot representation of GFP expression in human embryonic kidney 293T cells (HEK) transfected with GFPΔATG alone (donor) or with a mixture of GFPΔATG and either acceptorDR.1 or acceptorIR.1. Cultures cotransfected with GFPΔATG, acceptorScR and the I-SceI expression plasmid pCAG.I-SceI served as positive control. Flow cytometry was carried out 3 days post-transfection with 10 000 viable cells being analyzed per sample. (C) Live-cell imaging by phase-contrast and fluorescence microscopy of monolayers of African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorIR.1 or acceptorDR.1. Parallel cultures exposed to GFPΔATG, acceptorScR and pCAG.I-SceI served as positive control for HR-dependent GFP reconstitution. Microscopic analysis was performed 3 days post-transfection. Original magnification: ×40.
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gkr976-F5: Effect of repetitive DNA sequences in a direct or inverted repeat configuration on homology-directed gene repair in mammalian cells. (A) Flow cytometric analysis of human embryonic kidney 293T cells (HEK), human fetal retinoblasts (911 and PER.tTA.Cre76 [PER]) and African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorDR.1 or acceptorIR.1. Negative and positive controls for HR-mediated GFP repair in each of the tested cell types were provided by cells containing GFPΔATG and acceptorScR (−) or these two plasmids as well as pCAG.I-SceI (+), respectively. (B) Dot plot representation of GFP expression in human embryonic kidney 293T cells (HEK) transfected with GFPΔATG alone (donor) or with a mixture of GFPΔATG and either acceptorDR.1 or acceptorIR.1. Cultures cotransfected with GFPΔATG, acceptorScR and the I-SceI expression plasmid pCAG.I-SceI served as positive control. Flow cytometry was carried out 3 days post-transfection with 10 000 viable cells being analyzed per sample. (C) Live-cell imaging by phase-contrast and fluorescence microscopy of monolayers of African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorIR.1 or acceptorDR.1. Parallel cultures exposed to GFPΔATG, acceptorScR and pCAG.I-SceI served as positive control for HR-dependent GFP reconstitution. Microscopic analysis was performed 3 days post-transfection. Original magnification: ×40.

Mentions: Subsequently, we exposed cultures of HEK 293T cells, human fetal retinoblasts (911 and PER.tTA.Cre76 cells) and African green monkey kidney fibroblasts (COS-7 cells) to GFPΔATG and either acceptorDR.1 or acceptorIR.1. Again, negative and positive controls were provided by cotransfecting cultures of each of these cell types with a mixture of the donor plasmid GFPΔATG and acceptorScR alone or together with pCAG.I-SceI, respectively. Data depicted in Figure 5A show distinct levels of HR-dependent GFP repair in the various cell types tested. This might be the result of different transfection efficiencies and/or of intrinsic cell type-specific differences in the ability to recognize and process, via HR, DNA secondary structures. However, importantly, like in HeLa cells, appreciable HR-mediated GFP repair was only observed after cotransfection of GFPΔATG and acceptorIR.1 (Figure 5A). This IR.1-mediated HR stimulatory effect was independent of the amount of p53 present in the disparate cell types tested (Supplementary Figure S2). Some representative flow cytometry dot plots and direct fluorescence microscopy micrographs corresponding to these experiments are depicted in Figure 5B and C, respectively. Collectively, these experiments suggest that the inverted DNA repeat-induced HR is a mammalian cell type-independent phenomenon.Figure 5.


Nonspaced inverted DNA repeats are preferential targets for homology-directed gene repair in mammalian cells.

Holkers M, de Vries AA, Gonçalves MA - Nucleic Acids Res. (2011)

Effect of repetitive DNA sequences in a direct or inverted repeat configuration on homology-directed gene repair in mammalian cells. (A) Flow cytometric analysis of human embryonic kidney 293T cells (HEK), human fetal retinoblasts (911 and PER.tTA.Cre76 [PER]) and African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorDR.1 or acceptorIR.1. Negative and positive controls for HR-mediated GFP repair in each of the tested cell types were provided by cells containing GFPΔATG and acceptorScR (−) or these two plasmids as well as pCAG.I-SceI (+), respectively. (B) Dot plot representation of GFP expression in human embryonic kidney 293T cells (HEK) transfected with GFPΔATG alone (donor) or with a mixture of GFPΔATG and either acceptorDR.1 or acceptorIR.1. Cultures cotransfected with GFPΔATG, acceptorScR and the I-SceI expression plasmid pCAG.I-SceI served as positive control. Flow cytometry was carried out 3 days post-transfection with 10 000 viable cells being analyzed per sample. (C) Live-cell imaging by phase-contrast and fluorescence microscopy of monolayers of African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorIR.1 or acceptorDR.1. Parallel cultures exposed to GFPΔATG, acceptorScR and pCAG.I-SceI served as positive control for HR-dependent GFP reconstitution. Microscopic analysis was performed 3 days post-transfection. Original magnification: ×40.
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Related In: Results  -  Collection

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gkr976-F5: Effect of repetitive DNA sequences in a direct or inverted repeat configuration on homology-directed gene repair in mammalian cells. (A) Flow cytometric analysis of human embryonic kidney 293T cells (HEK), human fetal retinoblasts (911 and PER.tTA.Cre76 [PER]) and African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorDR.1 or acceptorIR.1. Negative and positive controls for HR-mediated GFP repair in each of the tested cell types were provided by cells containing GFPΔATG and acceptorScR (−) or these two plasmids as well as pCAG.I-SceI (+), respectively. (B) Dot plot representation of GFP expression in human embryonic kidney 293T cells (HEK) transfected with GFPΔATG alone (donor) or with a mixture of GFPΔATG and either acceptorDR.1 or acceptorIR.1. Cultures cotransfected with GFPΔATG, acceptorScR and the I-SceI expression plasmid pCAG.I-SceI served as positive control. Flow cytometry was carried out 3 days post-transfection with 10 000 viable cells being analyzed per sample. (C) Live-cell imaging by phase-contrast and fluorescence microscopy of monolayers of African green monkey kidney fibroblasts (COS-7) cotransfected with GFPΔATG and either acceptorIR.1 or acceptorDR.1. Parallel cultures exposed to GFPΔATG, acceptorScR and pCAG.I-SceI served as positive control for HR-dependent GFP reconstitution. Microscopic analysis was performed 3 days post-transfection. Original magnification: ×40.
Mentions: Subsequently, we exposed cultures of HEK 293T cells, human fetal retinoblasts (911 and PER.tTA.Cre76 cells) and African green monkey kidney fibroblasts (COS-7 cells) to GFPΔATG and either acceptorDR.1 or acceptorIR.1. Again, negative and positive controls were provided by cotransfecting cultures of each of these cell types with a mixture of the donor plasmid GFPΔATG and acceptorScR alone or together with pCAG.I-SceI, respectively. Data depicted in Figure 5A show distinct levels of HR-dependent GFP repair in the various cell types tested. This might be the result of different transfection efficiencies and/or of intrinsic cell type-specific differences in the ability to recognize and process, via HR, DNA secondary structures. However, importantly, like in HeLa cells, appreciable HR-mediated GFP repair was only observed after cotransfection of GFPΔATG and acceptorIR.1 (Figure 5A). This IR.1-mediated HR stimulatory effect was independent of the amount of p53 present in the disparate cell types tested (Supplementary Figure S2). Some representative flow cytometry dot plots and direct fluorescence microscopy micrographs corresponding to these experiments are depicted in Figure 5B and C, respectively. Collectively, these experiments suggest that the inverted DNA repeat-induced HR is a mammalian cell type-independent phenomenon.Figure 5.

Bottom Line: Despite their patent biological relevance, the parameters governing DNA repeat-associated chromosomal transactions remain ill-defined.We found that nonspaced DNA repeats can, per se, engage the HR pathway of the cell and that this process is primarily dependent on their spacing and relative arrangement (i.e. parallel or antiparallel) rather than on their sequence.Indeed, our data demonstrate that contrary to direct and spaced inverted repeats, nonspaced inverted repeats are intrinsically recombinogenic motifs in mammalian cells lending experimental support to their role in genome dynamics in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.

ABSTRACT
DNA repeats constitute potential sites for the nucleation of secondary structures such as hairpins and cruciforms. Studies performed mostly in bacteria and yeast showed that these noncanonical DNA structures are breakage-prone, making them candidate targets for cellular DNA repair pathways. Possible culprits for fragility at repetitive DNA sequences include replication and transcription as well as the action of structure-specific nucleases. Despite their patent biological relevance, the parameters governing DNA repeat-associated chromosomal transactions remain ill-defined. Here, we established an episomal recombination system based on donor and acceptor complementary DNA templates to investigate the role of direct and inverted DNA repeats in homologous recombination (HR) in mammalian cells. This system allowed us also to ascertain in a stringent manner the impact of repetitive sequence replication on homology-directed gene repair. We found that nonspaced DNA repeats can, per se, engage the HR pathway of the cell and that this process is primarily dependent on their spacing and relative arrangement (i.e. parallel or antiparallel) rather than on their sequence. Indeed, our data demonstrate that contrary to direct and spaced inverted repeats, nonspaced inverted repeats are intrinsically recombinogenic motifs in mammalian cells lending experimental support to their role in genome dynamics in higher eukaryotes.

Show MeSH