XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

Abstract

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to ends of DNA double-strand breaks and recruits several factors of the Non-Homologous End Joining (NHEJ) pathway through molecular mechanisms that remain unclear. Here, we describe the crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBMs motifs bind on remote sites of Ku80 α/β domain. The X-KBM occupies an internal pocket formed after an unprecedented large outward rotation of the Ku80 α/β domain. We reveal independent recruitment at laser-irradiated sites of the APLF-interacting protein XRCC4 and of XLF through the respective binding of A- and X-KBMs to Ku80. Finally, we show that mutations on the X-KBM and A KBM binding sites in Ku80 compromises efficiency and accuracy of end-joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchorage points necessary to build the NHEJ intricate interactions network.

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system

“…The protein KU70 acts during cNHEJ together with KU80 as a heterodimer that binds to broken DNA ends and recruits further repair proteins (Tamura et al ., ; Lieber, ). Recent results in mammals indicate that KU recruited proteins like XLF and APLF are responsible for keeping both broken ends in close proximity to avoid genomic instability (Downs and Jackson, ; Graham et al ., ; Nemoz et al ., ). The presence of the KU heterodimer seems to prevent MH‐mediated NHEJ factors like POLQ from processing DNA ends (Schimmel et al ., ).…”

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination

“…We propose instead that the Ku80c α/β domain specifically contributes to stabilizing Pgm on chromatin, either by interacting directly with a chromatin component or through a tri-partite interaction with another component of the excision complex or a yet unidentified chromatinassociated partner (Fig 7). The α/β domain of Ku80 (also called von Willebrand factor type A or vWA domain) was recently shown to interact with different partners, such as the accessory NHEJ factors APLF and XLF in humans [36,37] or the telomeric chromatin protein Sir4 in budding yeast [38], and may more generally act as a recruitment platform for Ku-interacting partners.…”

XLF acts as a flexible connector during non-homologous end joining

“…Collectively these results are consistent with the KBM playing a critical role in recruiting XLF to DSBs through its interaction with Ku. 25,26 The interior region of the C-terminal tail of XLF is essential for end joining.…”

“…FIG 4A) is likely because the Cterminal residues of the XLF KBM bind Ku80 in an internal hydrophobic pocket with the -COO group at the C-terminus forming an electrostatic contact with Lys238 of Ku80 (in the human). 26 The combination of losing this electrostatic contact and having to sterically accommodate the flexible linker within the hydrophobic pocket likely inhibits the KBM of subunit 1 from binding Ku80 effectively. A similar trend was observed when we used tdXLF constructs containing a previously characterized point mutation within the KBM (Leu 301 to Glu) (Supp.…”

“…25 , 26 Deletion of the KBM ablates cellular recruitment of XLF to sites of DNA damage 25 and mutations within the KBM result in varying defects in cellular end joining assays and cell survival in the presence of DSB inducing agents. 26,27 The contributions of the remainder of the C-terminal tail, henceforth referred to as the tail, are unknown, although it has been shown to be a target for phosphorylation by DNA-PKcs. 28 Across vertebrate species, there is poor sequence conservation within this region, and yet the tail is invariably retained with a similar length (Supp.…”