Anoxia Tolerant DNA Replication is Supported by ATR Kinase in the Annual Killifish Austrofundulus limnaeus

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G ₁ after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased γH2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G ₂ checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G ₁ phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.