Limits...
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.

Show MeSH
Testing the impact of target DNA synthesis on DNA repeat-mediated homology-directed gene repair. (A) SV40 ori-dependent DNA replication of acceptor constructs. Acceptor plasmids containing the test sequences ScR, IR.1, DR.1 or spIR.1 and with or without SV40 ori were transfected into COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. At 3 days post-transfection, extrachromosomal DNA was extracted and treated with XbaI and the prokaryotic DNA methylation pattern-sensitive restriction enzyme DpnI. After agarose gel electrophoresis, the resolved DNA was subjected to Southern blot analysis using a GFP-specific probe. DpnI-resistant, de novo replicated DNA, was detected only in samples of cells transfected with SV40 ori-positive acceptor plasmids (right-hand side upper panel). (B) Relative homology-directed gene repair frequencies in COS-7 cells transfected with GFPΔATG, the indicated acceptor plasmids with (+) or without (−) SV40 ori and in one case also pCAG.I-SceI.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3300023&req=5

gkr976-F7: Testing the impact of target DNA synthesis on DNA repeat-mediated homology-directed gene repair. (A) SV40 ori-dependent DNA replication of acceptor constructs. Acceptor plasmids containing the test sequences ScR, IR.1, DR.1 or spIR.1 and with or without SV40 ori were transfected into COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. At 3 days post-transfection, extrachromosomal DNA was extracted and treated with XbaI and the prokaryotic DNA methylation pattern-sensitive restriction enzyme DpnI. After agarose gel electrophoresis, the resolved DNA was subjected to Southern blot analysis using a GFP-specific probe. DpnI-resistant, de novo replicated DNA, was detected only in samples of cells transfected with SV40 ori-positive acceptor plasmids (right-hand side upper panel). (B) Relative homology-directed gene repair frequencies in COS-7 cells transfected with GFPΔATG, the indicated acceptor plasmids with (+) or without (−) SV40 ori and in one case also pCAG.I-SceI.

Mentions: SV40 is a mammalian double-stranded DNA virus with a circular genome whose replication has been extensively studied as a model for chromosomal nuclear DNA replication in higher eukaryotes (28). The only viral cis-acting element and trans-acting factor required for SV40-dependent DNA replication are the ori and the large T antigen protein, respectively. Thus, to investigate the impact of target template replication on DNA repeat-induced HR in mammalian cells, the SV40 ori was introduced at equivalent positions in acceptorScR, acceptorDR.1, acceptorIR.1 and acceptorspIR.1 to generate the constructs acceptorScR.ORI, acceptorDR.1.ORI, acceptorIR.1.ORI and acceptorspIR.1.ORI, respectively. Next, each of these plasmids was individually transfected into the SV40 large T-expressing COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. Extrachromosomal DNA isolated from these cells was treated with DpnI to selectively digest the input prokaryotic DNA and with XbaI to linearize de novo synthesized acceptor DNA molecules and HR products. Southern blot analysis of the digestion products using a GFP-specific probe revealed SV40 ori-dependent accumulation of de novo generated DNA molecules, demonstrating the replication proficiency of the SV40 ori-containing acceptor plasmids in COS-7 cells (Figure 7A). Interestingly, acceptor DNA replication did not lead to a significant increase in homology-directed gene repair levels in any of the experimental setups tested (Figure 7B). Taken together, these data indicate that under the prevailing experimental conditions, replication of acceptor DNA molecules carrying a palindrome, a direct repeat or a spaced inverted repeat does not significantly enhance homology-directed gene repair.Figure 7.


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)

Testing the impact of target DNA synthesis on DNA repeat-mediated homology-directed gene repair. (A) SV40 ori-dependent DNA replication of acceptor constructs. Acceptor plasmids containing the test sequences ScR, IR.1, DR.1 or spIR.1 and with or without SV40 ori were transfected into COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. At 3 days post-transfection, extrachromosomal DNA was extracted and treated with XbaI and the prokaryotic DNA methylation pattern-sensitive restriction enzyme DpnI. After agarose gel electrophoresis, the resolved DNA was subjected to Southern blot analysis using a GFP-specific probe. DpnI-resistant, de novo replicated DNA, was detected only in samples of cells transfected with SV40 ori-positive acceptor plasmids (right-hand side upper panel). (B) Relative homology-directed gene repair frequencies in COS-7 cells transfected with GFPΔATG, the indicated acceptor plasmids with (+) or without (−) SV40 ori and in one case also pCAG.I-SceI.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300023&req=5

gkr976-F7: Testing the impact of target DNA synthesis on DNA repeat-mediated homology-directed gene repair. (A) SV40 ori-dependent DNA replication of acceptor constructs. Acceptor plasmids containing the test sequences ScR, IR.1, DR.1 or spIR.1 and with or without SV40 ori were transfected into COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. At 3 days post-transfection, extrachromosomal DNA was extracted and treated with XbaI and the prokaryotic DNA methylation pattern-sensitive restriction enzyme DpnI. After agarose gel electrophoresis, the resolved DNA was subjected to Southern blot analysis using a GFP-specific probe. DpnI-resistant, de novo replicated DNA, was detected only in samples of cells transfected with SV40 ori-positive acceptor plasmids (right-hand side upper panel). (B) Relative homology-directed gene repair frequencies in COS-7 cells transfected with GFPΔATG, the indicated acceptor plasmids with (+) or without (−) SV40 ori and in one case also pCAG.I-SceI.
Mentions: SV40 is a mammalian double-stranded DNA virus with a circular genome whose replication has been extensively studied as a model for chromosomal nuclear DNA replication in higher eukaryotes (28). The only viral cis-acting element and trans-acting factor required for SV40-dependent DNA replication are the ori and the large T antigen protein, respectively. Thus, to investigate the impact of target template replication on DNA repeat-induced HR in mammalian cells, the SV40 ori was introduced at equivalent positions in acceptorScR, acceptorDR.1, acceptorIR.1 and acceptorspIR.1 to generate the constructs acceptorScR.ORI, acceptorDR.1.ORI, acceptorIR.1.ORI and acceptorspIR.1.ORI, respectively. Next, each of these plasmids was individually transfected into the SV40 large T-expressing COS-7 cells together with the homologous donor construct GFPΔATG or the nonhomologous donor plasmid RFPΔATG. Extrachromosomal DNA isolated from these cells was treated with DpnI to selectively digest the input prokaryotic DNA and with XbaI to linearize de novo synthesized acceptor DNA molecules and HR products. Southern blot analysis of the digestion products using a GFP-specific probe revealed SV40 ori-dependent accumulation of de novo generated DNA molecules, demonstrating the replication proficiency of the SV40 ori-containing acceptor plasmids in COS-7 cells (Figure 7A). Interestingly, acceptor DNA replication did not lead to a significant increase in homology-directed gene repair levels in any of the experimental setups tested (Figure 7B). Taken together, these data indicate that under the prevailing experimental conditions, replication of acceptor DNA molecules carrying a palindrome, a direct repeat or a spaced inverted repeat does not significantly enhance homology-directed gene repair.Figure 7.

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