Structural basis of long-range to short-range synaptic transition in NHEJ

Siyu Chen; Linda Lee; Tasmin Naila; Susan Fishbain; Annie Wang; Alan E. Tomkinson; Susan P. Lees-Miller; Yuan He

doi:10.1038/s41586-021-03458-7

Structural basis of long-range to short-range synaptic transition in NHEJ

Siyu Chen, Linda Lee, Tasmin Naila, Susan Fishbain, Annie Wang, Alan E. Tomkinson, Susan P. Lees-Miller, Yuan He^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

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Abstract

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis^1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors^2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.

Original language	English (US)
Pages (from-to)	294-298
Number of pages	5
Journal	Nature
Volume	593
Issue number	7858
DOIs	https://doi.org/10.1038/s41586-021-03458-7
State	Published - May 13 2021

Funding

Acknowledgements We thank J. Pattie for computer support, J. Meyers, R. M. Haynes and H. Scott at the PNCC for data collection support, D. Ramsden for the gift of a rabbit anti-phosphoT2609 reagent, A. Rosenzweig and I. Radhakrishnan for discussion and comments on the manuscript. This work was supported by a Cornew Innovation Award from the Chemistry of Life Processes Institute at Northwestern University (to Y.H.), a Catalyst Award by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (to Y.H.), an Institutional Research Grant from the American Cancer Society (IRG-15-173-21 to Y.H.), an H Foundation Core Facility Pilot Project Award (to Y.H.), a Pilot Project Award under U54CA193419 (to Y.H.) and NIH grant R01 GM135651 (to Y.H.). S.C. is supported by the Molecular Biophysics Training Program from NIGMS/NIH (5T32 GM008382). A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work used the Sapphire imager from the Northwestern University Keck Biophysics Facility funded by NIH grant 1S10OD026963-01, as well as the resources of the Northwestern University Structural Biology Facility, which is generously supported by NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. The Gatan K2 direct electron detector was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. Work in the Lees-Miller laboratory was supported by Canadian Institutes of Health grant 16939 and the Engineered Air Chair in Cancer Research. Work in the Tomkinson laboratory was supported by NIH grant R01GM047251, as well as the University of New Mexico Comprehensive Cancer Center supported by NCI CCSG P30 CA118100 grant. The collaboration between the He, Lees-Miller and Tomkinson laboratories was supported by NCI P01 CA092584.

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10.1038/s41586-021-03458-7

Cite this

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abstract = "DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 {\AA} apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.",

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AU - Lees-Miller, Susan P.

AU - He, Yuan

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Structural basis of long-range to short-range synaptic transition in NHEJ

Abstract

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NHEJ Short-range synaptic complex

NHEJ Long-range synaptic complex

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Structural basis of long-range to short-range synaptic transition in NHEJ

Abstract

Funding

ASJC Scopus subject areas

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NHEJ Short-range synaptic complex

NHEJ Long-range synaptic complex

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