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A Persistent Double-Strand Break Destabilizes Human DNA in Yeast and Can Lead to G₂ Arrest and Lethality¹

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709

Double-strand breaks (DSBs) are an important source of genomic change in many organisms. We have examined the consequences of a persistent versus a rapidly repaired DSB on cell progression, viability, and stability of human DNA contained in dispensable yeast artificial chromosomes (YACs) within the yeast Saccharomyces cerevisiae. An Alu-URA3-YZ integrating 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 identified previously: those that could be rapidly repaired (RR-DSB), through recombination between flanking Alus; and persistent DSBs (C. B. Bennett et al., Mol. Cell. Biol., 16: 4414–4425, 1996). These persistent DSBs (type 1) resulted in G₂ delay and lethality. A third class of DSB is now identified corresponding to a persistent DSB that does not lead to G₂ arrest or lethality (type 2). Unlike YACs in which the DSB was rapidly repaired, the two types of persistent DSBs destabilized the human YAC DNA, resulting in a high likelihood of YAC loss (85% of surviving colonies). Furthermore, both types of persistent DSBs could be misrepaired, resulting in mostly large internal or terminal deletions in the retained YACs. Therefore, recovery of these altered YACs can occur regardless of the effect of the DSBs on G₂ arrest and cell lethality. If similar events occur in mammalian cells, persistent DSBs could be the initiating events that lead to a loss of heterozygosity and the expression of recessive oncogenes seen in malignant cells.

¹ Partial support was provided by an interagency agreement from the Department of Energy DE-A105-94ER61940.

² To whom requests for reprints should be addressed. Phone: (919) 541-0972; Fax: (919) 541-7593; E-mail: bennett_c@niehs.nih.gov.

Received 7/31/96. Accepted 3/24/97.

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