High-throughput screening identifies a previously undescribed checkpoint controlling mitotic progression in response to DNA damage.

Abstract

Following DNA damage, the cell cycle can be slowed or halted to allow for DNA repair. However, the mechanisms underpinning mitotic delay in response to DNA damage are unclear. Through an unbiased high-throughput screen, here, we have identified superoxide dismutase 1 (SOD1) as an essential factor mediating mitotic delay in response to DNA damage. Cells with damaged DNA arrest at metaphase, indicating involvement of the spindle assembly checkpoint (SAC); however, this response is lost following SOD1 depletion. Furthermore, whilst depletion of SAC proteins promotes rapid cell division (often less than 10 min) in all conditions, SOD1 depletion has no impact on mitotic progression either in unperturbed mitosis or in response to spindle poisons and does not decrease the mitotic transit time beyond the normal rate. Cells depleted of SOD1 display damaged centromeres and mitotic defects but no longer exhibit DNA-damage-induced mitotic delay. SOD1 has previously been shown to mediate redox control of phosphatases such as PP2a. In response to DNA damage, we observed elevated phosphorylation of SAC protein BubR1 and the kinetochore protein KNL1. Dephosphorylation of these proteins is required for SAC silencing, and PP2a has previously been implicated in this. Following SOD1 depletion, we observed elevated PP2a activity and decreased phosphorylation of BubR1 and KNL1. We propose that, in response to damage, SOD1 restrains PP2a activity, resulting in elevated BubR1 and KNL1 phosphorylation leading to persistent SAC activation.