The nature of the DNA damage-induced
checkpoint signal that causes the arrest of budding yeast prior to
mitosis is unknown. To determine if this signal is transmitted through
the cytoplasm or is confined to the nucleus, we created binucleate
heterokaryon yeast cells in which one nucleus suffered an unrepairable
double-strand break caused by expression of the HO endonuclease, while
the second nucleus, lacking an HO cleavage site, was undamaged. The
damaged nucleus contained tandem repeats of the of Lac operator site
(lacO) providing binding sites for a GFP-lacI fusion
protein and making it possible to distinguish it from the undamaged
one. Without HO-induction (DNA damage) the two nuclei divided
synchronously in the majority of the cells. In contrast, when
DNA-damage was induced, in more than 90% of the cases the damaged
nucleus arrested prior to mitosis, while the undamaged nucleus
completed division. When HO endonuclease was expressed, but the two
nuclei were deleted for the RAD9 checkpoint gene, both nuclei
divided simultaneously. We made the DNA damage signal stronger by
increasing the number of double-strand breaks in the damaged nucleus
from one to two, or we made it more persistent by deleting
YKU70. In neither of these cases was the division of the
undamaged nucleus affected. These results suggest that in yeast the
DNA damage checkpoint causing G2/M arrest is regulated by a signal
that is nuclear-limited.
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