Structural studies of proteins involved in V(D)J recombination

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Our research program on the structural analysis of proteins involved in V(D)J recombination has continued in the past year. Our detailed structural dissection of all stages of DNA cleavage by the RAG1-RAG2 protein complex, which initiates this process, has now been completed, with two papers published in 2020. In continued collaboration with the group of Dr. Wei Yang, we have now moved on to the DNA-rejoining phase of V(D)J recombination, focusing on the DNA-dependent protein kinase (DNA-PK), the very large protein (4128 residues) that coordinates multiple steps of repair. In 2020, we obtained structures by cryoEM of this protein either alone or bound to DNA, or in complex with both its protein cofactor Ku70/80 and DNA, in several conformational states that govern its kinase activity. We now have high-resolution structures in the presence of ATP, and find forms of DNA-PK that either protect a DNA end or make it available for processing. In the second case, a gross rearrangement of the phosphorylated DNA-PK structure opens a wide space for access by Artemis, the enzyme that opens DNA hairpins, explaining why DNA-PK, but not Artemis, needs to be phosphorylated for hairpin processing to occur. As DNA-PK belongs to the PIKK kinase family that includes ATM, ATR, and the metabolic sensors SMG and TOR, such large changes in structure may be important for these kinases as well. This work has been submitted for publication.