Capture of DNA sequences at double-strand breaks in mammalian chromosomes.
To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we 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. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.
Lin
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Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA.
Waldman
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AS
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002875:Chromosomes / Q000648:ultrastructure*; D004249:DNA Damage; D004251:DNA Transposable Elements / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D029701:Saccharomyces cerevisiae Proteins; D013937:Thymidine Kinase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific