A PERSISTENT DOUBLE-STRAND BREAK DESTABILIZES HUMAN DNA IN YEAST AND CAN LEAD TO G(2) ARREST AND LETHALITY

Double-strand breaks (DSBs) are an important source of genomic change in many organisms. We have examined the consequences of a persistent v ersus a rapidly repaired DSB on cell progression, viability, and stabi lity of human DNA contained in dispensable yeast artificial chromosome s (YACs) within the yeast Saccharomyces cerevisiae, An Alu-URA3-YZ int egrating plasmid was used to target the YZ sequence to repetitive Alu sequences within the human YAC. The YZ site can be cut by an inducible HO-endonuclease resulting in a DSB, Two classes of DSBs had been iden tified previously: those that could be rapidly repaired (RR-DSB), thro ugh recombination between flanking Alus; and persistent DSBs (C. B. Be nnett et al., Mol. Cell. Biol., 16: 4114-4425, 1996). These persistent DSBs (type 1) resulted in G(2) delay and lethality, A third class of DSB is now identified corresponding to a persistent DSB that does not lead to G(2) arrest or lethality (type 2), Unlike YACs in which the DS B was rapidly repaired, the two types of persistent DSBs destabilized the human YAC DNA, resulting in a high likelihood of YAC loss (similar to 85% of surviving colonies), Furthermore, both types of persistent DSBs could be misrepaired, resulting in mostly large internal or termi nal deletions in the retained YACs. Therefore, recovery of these alter ed YACs can occur regardless of the effect of the DSBs on G(2) arrest and cell lethality, If similar events occur in mammalian cells, persis tent DSBs could be the initiating events that lead to a loss of hetero zygosity and the expression of recessive oncogenes seen in malignant c ells.