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

Michal Hammel; Yaping Yu; Sarvan K. Radhakrishnan; Chirayu Chokshi; Miaw-Sheue Tsai; Yoshihiro Matsumoto; Monica Kuzdovich; Soumya G. Remesh; Shujuan Fang; Alan E. Tomkinson; Susan P. Lees-Miller; John A. Tainer

doi:10.1074/jbc.M116.751867

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

From the ^‡Molecular Biophysics & Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720,
the ^§Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada,
the ^¶University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131, and
the ^‖Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

↵1 To whom correspondence may be addressed. E-mail: mhammel{at}lbl.gov.
↵2 To whom correspondence may be addressed. E-mail: leesmill{at}ucalgary.ca.
↵3 Supported by startup funds from the Cancer Prevention and Research Institute of Texas, and the University of Texas STARs program. To whom correspondence may be addressed. E-mail: JATainer{at}LBL.gov.

Edited by Patrick Sung

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.

Footnotes

↵* This work was supported in part by National Institutes of Health Structural Cell Biology of DNA Repair Machines P01 Grant CA92584 (to J. A. T., M. H., A. E. T., and S. P. L. M.), Canadian Institutes of Health Research operating Grant MOP13639 (to S. P. L. M.), the Robert A. Welch Distinguished Chair in Chemistry (to J. A. T.), the Engineered Air Chair in Cancer Research (to S. P. L. M.), and National Institutes of Health Grant R01 GM47251 (to A. E. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.