Structural studies of proteins involved in DNA repair and recombination

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Continuing our work on the initiation of V(D)J recombination by the RAG1/2 protein complex, we have obtained a more extended protein structure containing the full C-terminal of RAG2 in addition to the catalytically active parts of RAG1 and RAG2 previously reported. This C-terminal extension contains a PHD domain that binds to a particular histone modification: histone 3 modified by trimethylation on Lysine 4 (known as H3K4Me) that characterizes active chromatin. The PHD domain thus helps to bring RAG1/2 to its desired sites of action. In addition, the PHD domain, when free of histone, inhibits RAG1/2 cleavage of DNA. Thus, the PHD domain pays a dual role in regulating V(D)J recombination. Our new structure makes it clear how this inhibition occurs. Continuing our work on the DNA-dependent protein kinase (DNA-PK), we are now looking at its role in initiating homologous recombination. This is known to involve its interaction with the three-protein complex of Mre11, Rad50, and Nbs 1 (MRN), which has both endo- and exonuclease activity near a DNA-PK blocked DNA end, thus creating the necessary 3 overhang on DNA. This challenging multi-protein complex (seven proteins altogether, including cofactors) is being studied by cryo-electron microscopy.