Long inverted repeats are an at-risk motif for recombination in mammalian cells

Abstract

Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation. Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.