Abstract
Aprataxin polynucleotide kinase/phosphatase-like factor (APLF) facilitates nonhomologous end joining (NHEJ) and associates with the core NHEJ components XRCC4-DNA ligase IV and Ku. The APLF forkhead-associated (FHA) domain directs interactions with XRCC4, but the APLF-Ku interaction has not been well characterized. Here we describe an evolutionarily conserved amino acid motif within APLF that is required for mediating the physical interaction between APLF and Ku. This APLF Ku-binding motif possesses a similarity to regions identified in other NHEJ factors, WRN and XLF, which also direct interactions with Ku. Indeed, peptides derived from the Ku-binding region of APLF, WRN, or XLF were sufficient to reconstitute the interaction with Ku in vitro. Although APLF is localized predominantly to the nucleus, it does not possess a nuclear localization signal (NLS). Interestingly, the disruption of the APLF-Ku interaction by substituting key residues in the APLF Ku-binding motif was associated with increased relocalization of APLF to the cytoplasm and reduced association with XRCC4, which was rescued by the introduction of an NLS onto APLF. When human cells stably depleted of APLF were reconstituted with APLF Ku-binding mutants, or with an APLF FHA mutant that is known to disrupt interactions with XRCC4, APLF-dependent NHEJ and the retention of APLF at sites of laser-generated DNA damage were impaired. These data suggest functional requirements for Ku and XRCC4 in APLF-dependent NHEJ and a unique role for Ku as a factor required to facilitate the nuclear retention of APLF.