Chromosome healing in mouse embryonic stem cells

The addition of new telomeres to the ends of broken chromosomes, termed chr omosome healing, has been extensively studied in unicellular organisms; how ever, its role in the mammalian cell response to double-strand breaks is un known. A system for analysis of chromosome healing, which involves the inte gration of plasmid sequences immediately adjacent to a telomere, has been e stablished in mouse embryonic stem cells. This "marked" telomere contains a neo gene for positive selection in G418, an I-SceI endonuclease recognitio n sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells th at have lost the telomere. Transient expression of the I-SceI endonuclease results in terminal deletions involving telomeric repeat sequences added di rectly onto the end of the broken chromosome, The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3' ov erhang generated by the I-SceI endonuclease, without the loss of a single n ucleotide front the end of the chromosome. The next most frequent site invo lved 5 bp of complementarity, which occurred after the loss of four nucleot ides from the end of the chromosome. The new telomeres are generally much s horter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanis m for repair of chromosome breaks in mammalian cells.