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 and blocks DNA transposition by RAG1/2. Thus, the PHD domain pays a dual role in regulating V(D)J recombination. Our new structure makes it clear how this inhibition occurs. This structural work has now been refined to a higher level of resolution and published. It clarifies how RAG1/2, though derived from transposons in more primitive organisms, has evolved to carry out DNA cleavage without allowing transposition that would be harmful in the immune system. Moving on the last stage of non-homologous end-joining (NHEJ), we have completed and refined new structural and biochemical information on the end-filling and end-joining complex, an intricate complex of 7 distinct proteins (16 protein units altogether). This work shows how DNA polymerase Mu and DNA ligase 4 collaborate to fill in any gaps in DNA ends before ends are joined. It clarifies the detailed role of the cofactors Ku, XRCC4, XLF, and PAXX in holding the structural framework, and adjusting its spacing for joining to occur. The structure also indicates possible target sites for chemotherapy to inhibit NHEJ in treating cancer. This work has been published. Continuing our work on the DNA-dependent protein kinase (DNA-PK), we are now looking at its role in initiating homologous recombination when DNA ends are unsuitable for NHEJ.. In this process, DNA-PK interacts with the three-protein complex of Mre11, Rad50, and Nbs1 (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 biochemical and structural methods.