Abstract
The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.
Highlights
Double-strand breaks (DSBs) are the most deleterious DNA lesions and are caused by endogenous reactive oxygen species derived from cell metabolism, as well as by exogenous agents such as ionising radiation (IR)
We demonstrate that Nucleoporin 54 (Nup54) depletion increases IR-induced cell death by mitotic catastrophe, and enhances both the length of the G2 arrest and the sensitivity of cells irradiated in S and G2 phases, as well as the formation of chromosome aberrations arising from post-replicative DNA DSBs
The results presented here so far suggest that Nup54 KD causes an effect on cell cycle progression and sensitivity of cells irradiated in S and G2 phases, which might reflect the increased mitotic catastrophe observed by time lapsemicroscopy
Summary
Double-strand breaks (DSBs) are the most deleterious DNA lesions and are caused by endogenous reactive oxygen species derived from cell metabolism, as well as by exogenous agents such as ionising radiation (IR). Cells can undergo cell death, typically by mitotic catastrophe, or can survive and transmit the genetic alterations to their progeny, eventually leading to pathological conditions such as cancer [2]. The lethal effect that DSBs can have on cells is exploited in many cancer therapies, with radiotherapy being the most representative example. It is recognized that the capability of cancer cells to repair DSBs and/or prevent mitotic catastrophe, i.e. intrinsic radiosensitivity, is a major limitation for radiotherapy [4]. Understanding the mechanisms whereby cells deal with and survive DSBs is important for manipulating intrinsic radiosensitivity and improving radiotherapy
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