Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

Michal Hammel,Yaping Yu,Brandi L Mahaney,Brandon Cai,Ruiqiong Ye,Barry M Phipps,Robert P Rambo,Greg L Hura,Martin Pelikan,Sairei So,Ramin M Abolfath,David J Chen,Susan P Lees-Miller,John A Tainer

doi:10.1074/jbc.m109.065615

Abstract

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.

Highlights

DNA double strand breaks (DSBs)4 are among the most cytotoxic forms of DNA damage [1]
DNA-PKcs, DNA-dependent protein kinase catalytic subunit; SAXS, small angle x-ray scattering; MES, minimal ensemble search; double-stranded DNA (dsDNA), doublestranded DNA; SEC-MALS, size exclusion chromatography equipped with a multiangle light scattering detector; FRAP, fluorescence recovery after photobleaching; MD, molecular dynamics; EM, electron microscopy; Ku80CTR, Ku80 C-terminal region; Y-DNA, dsDNA with a Y-shaped structure at one end; HP-DNA, duplex with a hairpin at one end; AMP-PNP, 5Ј-adenylyl-␤,␥-imidodiphosphate
The SAXS profile and linear radius of gyration (RG) of the free Ku heterodimer over a concentration range of 1–10 mg/ml indicated that the protein is well behaved over a wide concentration range and adopts an aggregation-free state in solution (Fig. 1A and supplemental Fig. S1A)

Summary

EXPERIMENTAL PROCEDURES

Data Collection and Evaluation—SAXS data were collected at the ALS beamline 12.3.1 (Lawrence Berkeley National Laboratory, Berkeley, CA) [20]. Tunable wavelength ␭ 1.0 –1.5 Å and the sample-to-detector distances were set to 1.5 m, resulting in scattering vectors, q, ranging from 0.007 to 0.31 ÅϪ1. The experimental SAXS data for different protein concentrations were investigated for aggregation using Guinier plots [21]. The program GNOM [22] was used to compute the pair distance distribution functions, P(r). This approach provided the maximum dimension of the macromolecule, Dmax. The overall shapes were restored from the experimental data using the program DAMMIN [23] or GASBOR [24], respectively. Other Methods—Experimental procedures for protein purification, cell culture and transfection, live cell imaging, biotin pull-down assays, and limited proteolysis assays are provided in the supplemental material

RESULTS

To examine the flexibility of the

DISCUSSION