The non-homologous end-joining (NHEJ) pathway is one of two pathways responsible for repairing double-stranded breaks in cellular DNA. Double-stranded breaks can be formed by ionizing radiation or mutagenic chemicals, or as intermediates during normal processes such as the recombination of DNA segments required to create antibody and T cell receptor diversity. In contrast to homologous recombination, in which a double-stranded break is repaired by lining it up with a homologous piece of DNA, NHEJ can ligate any two free pieces of DNA together without requiring extensive sequence homology. Several proteins are known to be involved in NHEJ, but the details of how the complex of these proteins with DNA brings DNA ends together for ligation has been unknown.
Image of PDB ID 7LSY, Chen S, Lee L, Naila T, et al. Structural basis of long-range to short-range synaptic transition in NHEJ. Nature. 2021;78: 2179-2183, created using Mol* D. Sehnal, A.S. Rose, J. Kovca, S.K. Burley, S. Velankar (2018) Mol*: Towards a common library and tools for web molecular graphics MolVA/EuroVis Proceedings. doi:10.2312/molva.20181103), at the RCSB PDB (rcsb.org), H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242.
In a new publication in Nature, Chen et al characterize the structure of the protein-DNA complexes involved in NHEJ, and propose a mechanism by which the ends of two DNA molecules are captured by a large complex of proteins and then brought together in a short-range synaptic complex in which the ends are aligned for ligation to close the break. The authors used single particle cryo-electron microscopy to visualize the protein-DNA complexes.
The authors formed the long-range complex by first incubating DNA with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) followed by the addition of other complex components LigIV-XRCC4 and XLF. Their analysis demonstrated that the resulting 1.66 megadalton structure consists of two complexes of DNA-PK and LigIV-RXCC4, each bound to a DNA end, connected in the middle by one homodimer of XLF. In this complex, the two DNA ends are about 115 angstroms apart, not close enough for ligation.
Comparing the structure to other protein kinases, the authors predicted that the two DNA-PK molecules in the long-range complex are in an active state, and hypothesized that autophosphorylation of each DNA-PK by the other could act as a switch, releasing DNA-PK from the complex. Therefore, they formed a short-range synaptic complex including all of the proteins used earlier except DNA-PKcs. Their structural analyses showed that the resulting complex had a similar overall architecture to the long-range complex but with components shifted and rotated in such a way as to align the DNA ends perfectly for ligation.
The predictions were validated by carrying out in vitro ligation assays in which the Cy5-labeled ligation products were visualized using a Sapphire Biomolecular Imager. No ligation occurred in the long-range synaptic complex without the addition of ATP, an expected result since ATP would be required for the predicted transition to the short-range complex. In contrast, the short-range complex was found to readily ligate DNA, with both strands being ligated more often than single strands.
This important study provides insight into an essential cellular process, and analysis of interactions between components of the NHEJ complexes revealed by the structural study indicate potential mechanisms for some known pathologic mutations and could provide new targets for cancer therapy.
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