An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

Abstract

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…We describe factors affecting MRN functions at DSBs, stalled RFs, dysfunctional telomeres, and complexes combating exogenous viral DNA infection. We discuss how MRN acts more as a dynamic molecular machine for the expeditious formation of protein–nucleic acid functional scaffolds at DSBs, RFs, dysfunctional telomeres, and viral DNA than as part of linear pathways, as also proposed for nonhomologous end joining (NHEJ) complexes (24). As a multifunctional macromolecular machine, the structural biochemistry of the MRN complex initiates sensing of stalled forks and DSBs, cell cycle checkpoint signaling cascades, incision for and commitment to break repair pathways, reestablishment of posttranslational phosphorylations via ATM kinase, and functional regulation of chromatin remodeling.…”

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

“…This interaction has been mapped to the periphery of the Ku80 von Willebrand A domain (vWA) 5 . Ku-APLF interaction was shown to facilitate recruitment of the APLF-partner XRCC4 at damaged sites 9 and was proposed to stabilize the assembly of NHEJ factors around the DSB 19 . Notably, an A-KBM-like domain is present at the N-terminus of a recently identified inhibitor of the NHEJ pathway, CYREN(MRI), that also interacts with Ku80 20 (Figure 1a).…”

Genetic Single Nucleotide Polymorphisms (GSNPs) in the DNA Repair Genes and Hepatocellular Carcinoma Related to Aflatoxin B1 among Guangxiese Population

“…This gene is a generally expressed protein of 334 amino acids residues and functions together with DNA ligase IV and DNA-PK in the repair of DNA double-strand breaks. XRCC4 plays an important role in both nonhomologous end joining and the completion of V(D)J recombination [95,96]. Increasing evidence has shown that mutations in XRCC4 can cause short stature, microcephaly, and endocrine dysfunction (SSMED) resulting from more-deicient NHEJ capacity [97,98].…”