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Genetics, Vol. 158, 1665-1674, August 2001, Copyright © 2001

Capture of DNA Sequences at Double-Strand Breaks in Mammalian Chromosomes

Yunfu Lina and Alan S. Waldmana
a Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208

Corresponding author: Alan S. Waldman, Department of Biological Sciences, University of South Carolina, 700 Sumter St., Columbia, SC 29208., awaldman{at}sc.edu (E-mail)

Communicating editor: M. LICHTEN

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, ~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.





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