The PARP3- and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair.

APLF is a forkhead associated-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser(116)). Here, we demonstrate that the phosphorylation of APLF at Ser(116) in human U2OS cells by ATM is dependent on poly(ADP-ribose) polymerase 3 (PARP3) levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser(116) to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the level of accumulation of APLF at sites of laser-induced DNA damage and impaired the accumulation of Ser(116)-phosphorylated APLF at IR-induced γH2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signalling pathway to facilitate APLF-Ser(116) phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that Ser(116)-APLF phosphorylation facilitates APLF-dependent double-strand break repair.

A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization.

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity.

APLF (C2orf13) facilitates nonhomologous end-joining and undergoes ATM-dependent hyperphosphorylation following ionizing radiation.

Nonhomologous end-joining (NHEJ) is the major mammalian DNA double-strand break (DSB) repair pathway of DSBs induced by DNA damaging agents. NHEJ is initiated by the recognition of DSBs by the DNA end-binding heterodimer, Ku, and the final step of DNA end-joining is accomplished by the XRCC4-DNA ligase IV complex. We demonstrate that Aprataxin and PNK-like factor (APLF), an endo/exonuclease with an FHA domain and unique zinc fingers (ZFs), interacts with both Ku and XRCC4-DNA ligase IV in human cells. The interaction of APLF with XRCC4-DNA ligase IV is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. In contrast, APLF associates with Ku independently of the FHA and ZF domains, and APLF complexes with Ku at DNA ends. APLF undergoes ionizing radiation (IR) induced ATM-dependent hyperphosphorylation at serine residue 116, which is highly conserved across mammalian APLF homologues. We demonstrate further that depletion of APLF in human cells by siRNA is associated with impaired NHEJ. Collectively, these results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and XRCC4-DNA ligase IV.