DNA-PKcs-Dependent Signaling of DNA Damage in Dictyostelium discoideum

Abstract

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.

DNA Damage Signalling and Repair in Dictyostelium discoideum

“…Initially, Block et al and Hudson et al identified putative orthologues of Ku70 and Ku80 in the Dictyostelium genome. 49,50 Notably, Dictyostelium Ku80 contains a conserved C-terminal domain that is apparent only in organisms that possess DNA-PKcs and that is required to recruit this protein to sites of DNA damage. 51 These observations lead to speculation that Dictyostelium might contain an orthologue of DNA-PKcs and consistent with this hypothesis, an open reading frame that bares homology to this protein has been identified in the genome of this organism.…”

“…51 These observations lead to speculation that Dictyostelium might contain an orthologue of DNA-PKcs and consistent with this hypothesis, an open reading frame that bares homology to this protein has been identified in the genome of this organism. 49,50 Putative orthologues of the core NHEJ machinery, including DNA Ligase IV, XRCC4 and Artemis are also present in the Dictyostelium genome. 49 The identification of Figure 2.…”

“…50 Although disruption of the ku80 or dnapkcs genes does not result in any gross sensitivity to the DNA DSB inducing agent bleomycin during vegetative cell growth, these genes are required for the survival of Dictyostelium when bleomycin is administered to cells shortly after cells germinate from spores. This observation might be explained by a recent report suggesting that Dictyostelium cells arrest in G 1 -phase of the cell cycle during spore formation.…”

“…However, although DNA-PKcs contributes towards phosphorylation of H2A.X in higher eukaryotes, 62 neither the dnapkcs nor ku80 genes are required for phosphorylation of H2A.X in response to DNA DSBs during vegetative cell growth. 50 Although the kinase that phosphorylates H2A.X in vegatively growing Dictyostelium cultures remains to be identified, two possible candidates are the DNA-PKcs-related phosphatidylinositol-3-kinase-like kinases (PIKKs) ATM and ATR. The observation that Dictyostelium H2A.X phosphorylation in vegetative cell cultures is sensitive to the PIKK family inhibitor and radio-sensitising agent caffeine (D.-W. Hsu unpublished observations), might support this hypothesis.…”

“…63 Although an orthologue of ATM has yet to be identified in Dictyostelium, a putative orthologue of ATR exists in the genome of this organism. 50 ATR has been implicated in detecting DNA damage during S-phase, 64 in addition to the repair of DNA DSBs by HR. 65 Given that the majority of cells are in S or G 2 phase of the cell cycle in vegetative Dictyostelium cultures, 57 it is possible that ATR mediates phosphorylation of H2A.X under these growth conditions.…”