Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA.
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 &phi;X174 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 &phi;X174 DNA. Microhomology existed at most junctions between &phi;X174 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.
Lin
Y
Y
Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA.
Waldman
A S
AS
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002460:Cell Line; D019457:Chromosome Breakage; D002999:Clone Cells; D004247:DNA / Q000235:genetics; D004260:DNA Repair; D004278:DNA, Superhelical / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D017353:Gene Deletion; D051379:Mice; D016254:Mutagenesis, Insertional; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013936:Thymidine / Q000031:analogs & derivatives; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D016662:Virus Integration
D004278:DNA, Superhelical; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D013937:Thymidine Kinase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D013936:Thymidine