A DOUBLE-STRAND BREAK WITHIN A YEAST ARTIFICIAL CHROMOSOME (YAC) CONTAINING HUMAN DNA CAN RESULT IN YAC LOSS, DELETION, OR CELL LETHALITY

Human chromosomal DNA contains many repeats which might provide opport unities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12), An Alu-UR43-YZ sequenc e was targeted to several Alu sites within the YAC in strains of the y east Saccharomyces cerevisiae; the strains contained a galactose-induc ible HO endonuclease that cut the YAC at the YZ site, The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retentio n of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G(2) de lay associated with the persistent DSBs. The presence of persistent DS Bs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pul lback procedure, cell death was observed to begin at least 6 h after i nduction of a break, For YACs in which the DSB was rapidly repaired, t he breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous Conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl, Acad. Sci. USA 90 :5613-5617, 1993). However, a break in the YZ-CEN plasmid did not indu ce lethality in the strain (CBY) background used in the present study, The differences in survival levels appear to be due to the rapid degr adation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be l ong-lived.