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Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA
1Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA.
Abstract:
To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected. In a previous study of DSB-induced tk-deficient clones, we found that approximately 8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site. Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements. To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of φX174 genomic DNA. We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of φX174 DNA. Microhomology existed at most junctions between φX174 DNA and genomic sequences. Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome.
Insights
Mammalian cells can capture and integrate various DNA fragments into double-strand break (DSB) sites. This study shows that even random DNA fragments, like those from bacteriophage phiX174, can be recruited to repair DSBs.
Area of Science:
- Genetics
- Molecular Biology
- Genomics
Background:
- Double-strand breaks (DSBs) are critical DNA lesions that can lead to mutations.
- Understanding the mechanisms of DSB repair is crucial for genome stability.
- Previous studies indicated DNA fragment capture during DSB repair.
Purpose of the Study:
- To investigate whether specific DNA sequences are preferentially captured during DSB repair.
- To determine the capacity of mammalian cells to integrate exogenous DNA fragments at DSB sites.
Main Methods:
- Transfection of mouse fibroblasts with a thymidine kinase (tk) gene containing an I-SceI recognition site.
- Induction of DSBs using the I-SceI endonuclease.
- Electroporation with an I-SceI expression plasmid and bacteriophage phiX174 DNA fragments.
- Selection and analysis of tk-deficient clones to identify DNA capture events.
Main Results:
- 18 out of 132 double-strand break-induced tk-deficient clones captured DNA fragments.
- 14 of these events involved the capture of bacteriophage phiX174 DNA fragments.
- Microhomology was observed at most junctions between captured phiX174 DNA and genomic sequences.
Conclusions:
- Mammalian cells can efficiently capture and integrate various extrachromosomal DNA fragments at DSB sites.
- The process appears to favor integration at sites with microhomology.
- This suggests a broad capacity for DNA repair pathway recruitment in the mammalian genome.