The fidelity of cell division and development require genomic stability. Conserved signal transduction pathways called DNA structure dependent checkpoints help ensure genomic stability by detecting unreplicated or damaged DNA. Once detected, the pathways initiate responses that coordinate cell cycle progression with DNA repair processes, maintain telomere structure, induce cellular senescence or cause apoptosis [1, 2].
Members of the PI-3 kinase related kinase (PIKK) family are central to DNA structure dependent checkpoints and other stress-responsive pathways . PIKKs are large (>200 kD) proteins that harbor protein kinase activity in a conserved C-terminal catalytic domain that resembles the lipid kinase domain of PI-3 kinases. N-terminal to this kinase domain are protein-interaction and intramolecular folding domains. Following detection of a stress signal, changes in PIKK-protein interactions, folding and subcellular localization allow PIKKs to target downstream effector proteins and coordinate stress responses.
In fission yeast, a PIKK called Rad3 is central to DNA structure dependent checkpoints . Rad3 physically binds to Rad26, a regulatory subunit required for normal levels of Rad3-kinase activity [5, 6]. This Rad3/26 checkpoint complex is conserved throughout evolution and exists in humans (ATR/ATRIP), budding yeast (MECl/LCDlDDC2/PIE1), Xenopus (xATR/xATRIP) and possibly filamentous fungi (UvsB/UvsD) [7–12].
These Rad3/26 complexes are sensors that detect and respond to DNA structure checkpoint signals such as double-stranded breaks (DSBs) . Other conserved sensor complexes include the 9-1-1 (Rad9-Radl-Husl) complex and Crb2 [14–20]. The 9-1-1 complex appears to form a PCNA-like clamp that requires Radl7, a dynamic subunit of Replication Factor C, for loading onto DNA. Crb2 contains tandem BRCT-domains and resembles budding yeast Rad9 and human p53BPl. Following DNA damage, these three sensors relocalize independently of each other, suggesting that they detect aberrant DNA structures using parallel pathways [14, 21–23]. Exactly how the 9-1-1 and Rad3/26-like complexes initially detect damage is not well understood. They may recognize many different signals, including single-stranded DNA overhangs bound by single-stranded binding protein, and DNA damaged-induced changes in chromatin structure [24, 25]. Recent data suggest that the checkpoint signal for Crb2 localization is formed when DSBs alter the structure of nearby histones, and results obtained with p53BPl corroborate this finding [15, 26]. Following the production of checkpoint signals and their detection, the events leading to Rad3/26 kinase activation and downstream signal transduction require all three sensor complexes.
Depending on the checkpoint signal, the checkpoint-activated Rad3/26 kinase phosphorylates effector kinases Chkl or Cdsl, which in turn phosphorylate Mikl and Cdc25 . This leads to increased levels of Mikl, a negative Cdc2 regulator, and possibly reduces the phosphatase activity of Cdc25, a positive Cdc2 regulator [28–32]. Checkpoint regulation of Cdc25 may also be mediated by the fission yeast 14-3-3 proteins Rad24 and, to a lesser extent, Rad25 [32, 33]. These interactions compartmentalize Cdc25 in the cytoplasm, although the outcome of this is not understood . Recently, it was shown that Rad24 promotes checkpoint-dependent retention of Chkl in the nucleus . Therefore, 14-3-3 proteins may mediate the checkpoint response by affecting the localization of signaling proteins and checkpoint-targets. Interestingly, Rad24 is also required for proper cell morphogenesis, suggesting that this 14-3-3 protein is a component of pathways controlling cell shape .
We have been investigating why loss of rad26+ sensitizes cells to the microtubule depolymerizing agent thiabendazole (TBZ) . Specifically, we found that rad26Δ, rad3Δ, rad1Δ and rad9Δ cells were sensitive to TBZ, while hus1Δ and rad17Δ cells shared wild type TBZ-sensitivity. Therefore, TBZ sensitivity does not result from a defective DNA structure checkpoint.
The Mad2-dependent spindle assembly checkpoint restrains metaphase-to-anaphase progression when microtubules are compromised . Experiments have shown that overlap between the spindle assembly and DNA structure checkpoints exist. For example, the spindle assembly checkpoint of fission and budding yeast delays mitotic progression when DNA structure checkpoint mutants are treated with replication inhibitors [37–39]. Thus, the two checkpoint systems cooperate to enhance survival following genotoxic stress. Elements of these pathways may also cooperate to promote mitotic arrest following microtubule stress, which would explain why mutations in some fission yeast DNA structure checkpoint genes cause TBZ sensitivity.
Here, we initiated experiments to characterize the TBZ-sensitivity of rad26Δ cells. Our data show that rad26+ is required for the efficiency of two microtubule-dependent processes, chromosome segregation and cell polarity, and we suspect that defects in both processes may contribute to rad26Δ TBZ-sensitivity. Our data strongly suggest that Rad26 operates independently of the spindle assembly checkpoint to preserve both processes. With regard to the cell polarity defects of rad26Δ cells, our data show that rad26+ is required for proper growth patterns and the polar distribution of actin patches.
We also observed that microtubule-destabilizing conditions caused Rad26-GFP to accumulate in the cytoplasm by a Rad24-dependent manner. Possible outcomes of this response are discussed.