Mitotic catastrophe induced by overexpression of budding yeast Rad2p

Abstract

Mitotic catastrophe provokes endopolyploidy, giant cell formation and, eventually, delayed cell death. Mitotic catastrophe is induced by defective cell cycle checkpoints and by some anticancer drugs, ionizing radiation and microtubule-destabilizing agents. RAD2 is a yeast homologue of XPG, which is a human endonuclease involved in nucleotide excision repair. Here we show that Rad2p overexpression alone, in the absence of extrinsic DNA damage, causes cell growth arrest and mitotic catastrophe. Interestingly, Rad2p-induced cell growth arrest is not caused by the catalytic activity of Rad2p but rather by its C-terminal region. Cells growth-arrested by Rad2p induction do not show apoptotic phenotypes and deletion of YCA1, a yeast caspase homologue, does not affect cell growth arrest by Rad2p induction. However, Rad2p-induced cell growth arrest is released by rad9 deletion but is not affected by downstream DNA damage checkpoint genes. These observations suggest that RAD2 has a function in coordinating cell cycle regulation and damaged DNA repair.

Restoration of proliferation ability with increased genomic instability from Rad2p-induced mitotic catastrophe in Saccharomyces cerevisiae

“…As observed by the growth patterns on plates 26 , Rad2p over-expression completely inhibited cell growth for up to 40 hours in liquid medium ( Figure 1A). However, Rad2p-expressing cells resumed growth after 40 hours of induction and eventually reached a stationary stage similar to the vector control indicating that some of the cells escaped from growth arrest and regained the ability to duplicate.…”

“…Furthermore, Rad2p-induced cells displayed a morphology characteristic of mitotic catastrophe, and a significant portion of the cell population contained ¤2-fold the normal DNA content 26 . On the other hand, we observed that cells growth-arrested by Rad2p resumed growth after a prolonged incubation in galactose liquid medium, indicating that some cells experiencing Rad2p-induced mitotic catastrophe resumed cell growth.…”

“…Although it is possible that the re-growth was the result of small cells that remained haploid and took over the entire population, we excluded that possibility because the population would take less time to reach stationary stage if there were some small cells that were not affected by Rad2p expression. We rather deduced that the normal sized cells in Rad2p expressing cell cultures were the result of cells that experienced cell cycle arrest earlier than a majority of the cells in the population, because we used single colony based culture for all the experiments and Rad2p expression is leaky 26 .…”

“…This is because the cells were cultured in liquid medium on a Petri-dish without shaking in order to track budding patterns without causing separation of daughter cells from mother cells, and the cells were then photographed with an inverted microscope. However, liquid culture shaking did not lead to cellular aggregation 26 . Thus, the increased DNA content in Figure 1B and the cell mass in Figure 2A While the large buds were polyploid, like their parental cells, the small normal-sized buds from the enlarged parental cells were speculated to be haploid as the result of depolyploidization.…”

“…Interestingly, Rad2p-induced growth inhibition is due to neither increased apoptosis nor increased endonuclease activity of Rad2p. Instead, the C-terminal region of Rad2p is important for cell growth arrest 26 . These results suggest that Rad2p functions in cell cycle regulation through its C-terminal region.…”