SIR Functions Are Required for the Toleration of an Unrepaired Double-Strand Break in a Dispensable Yeast Chromosome

Abstract

Unrepaired DNA double-strand breaks (DSBs) typically result in G₂ arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G₂ arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G₂. There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G₂ checkpoint and high viability were restored in sir⁻ strains containing additional deletions of the silent mating type loci HMLand HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression ofMATa1 and MATα2 in Sir⁺ haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir⁺ function.

ACKNOWLEDGMENTS

We thank Virginia Zakian, Jeffrey Strathern, Kurt Runge, Kevin Lewis, and Dan Gietz for strains and plasmids and Hiep Tran for sequencing plasmid p54-45. We thank Kerry Bloom, Jim Mason, Dmitry Gordenin, Jake Kirchner, Kevin Lewis, Beverly Errede, and Doug Thrower for reviewing the manuscript and helpful discussions. C.B.B. also thanks D. Downie and C. Whatley for their technical expertise, without which this paper could not have been completed.

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