Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM

Michal Hammel; Daniel J Rosenberg; Jan Bierma; Gregory L Hura; Roopa Thapar; Susan P Lees-Miller; John A Tainer

doi:10.1016/j.pbiomolbio.2020.09.003

Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM

Prog Biophys Mol Biol. 2021 Aug:163:74-86. doi: 10.1016/j.pbiomolbio.2020.09.003. Epub 2020 Sep 20.

Authors

Michal Hammel¹, Daniel J Rosenberg², Jan Bierma³, Gregory L Hura³, Roopa Thapar⁴, Susan P Lees-Miller⁵, John A Tainer⁶

Affiliations

¹ Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. Electronic address: mhammel@lbl.gov.
² Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA; Graduate Group in Biophysics, University of California, Berkeley, CA, 94720, USA.
³ Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
⁴ Department of Cancer Biology, Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
⁵ Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, T2N 4N1, Canada.
⁶ Department of Cancer Biology, Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. Electronic address: JTainer@mdanderson.org.

Abstract

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

Keywords: Cryo-electron microscopy; DNA break Repair; Genome stability; Structural dynamics; X-ray scattering.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cryoelectron Microscopy
DNA
DNA Repair*
DNA-Activated Protein Kinase* / metabolism
Holoenzymes
Ku Autoantigen / metabolism
Scattering, Small Angle
X-Ray Diffraction

Substances

Holoenzymes
DNA
DNA-Activated Protein Kinase
Ku Autoantigen

Abstract

Publication types

MeSH terms

Substances

Grants and funding