RAD18 activates the G2/M checkpoint through DNA damage signaling to maintain genome integrity after ionizing radiation exposure.

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure

Depletion of RAD18 suppressed entry into the M phase from the G2 phase after IR exposure.

<p>(A) HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 1 or 2 Gy IR, and then lysed at the indicated time points, after irradiation. Samples were analyzed by western blotting with the indicated antibodies. (B) Cells were exposed to 1 Gy of IR, fixed with ethanol at the indicated time points after irradiation, and then immunostained with phospho-histone H3 and propidium iodide (PI). The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments. (C) Cells were exposed to various doses of IR and then fixed with ethanol 60 min after irradiation. The fixed cells were immunostained with phosphor-histone H3 and PI. The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments.</p

RAD18-deficiency increased apoptosis in murine thymocytes <i>in vivo</i>.

<p><i>Rad18</i>^<i>+/+</i>, <i>Rad18</i>^<i>+/-</i>, and <i>Rad18</i>^-/- mice were irradiated with 1Gy IR. Thymocytes were isolated at the time points indicated after irradiation. Apoptotic cell distributions in thymocytes were detected by using PE Annexin V Apoptosis Detection kit I and analyzed using flow cytometry. Each value represents the mean (+standard deviation) of the results from the individual mice.</p

Depleting RAD18 suppressed the response to DNA damage induced by IR.

<p>HEK293 cells were transfected with si-ctrl or si-RAD18, irradiated with 2 or 4 Gy, and then lysed at the indicated time points after irradiation. Samples were analyzed by western blotting with the indicated antibodies.</p