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Martin Gellert, Dale A. Ramsden, Tanya T. Paull, Kevin Hiom, Meni Melek, Mauro Modesti, *Dick C. van Gent
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
Bethesda, Maryland
*Erasmus University
Rotterdam, The Netherlands

Early and Late Steps in V(D)J Recombination

V(D)J recombination is known to occur in two very distinct stages. In the first stage, the RAG1 and RAG2 proteins act together to make DNA double-strand breaks at the recombination signal sequences (RSSs). Each break generates a blunt end (signal end) on the RSS and a DNA hairpin (coding end) on adjacent DNA that codes for the antigen receptor. In the second stage, these double strand breaks are resolved by joining the coding ends to produce the mature antigen receptor gene. The signal ends are also joined together in a separate reaction. The joining reactions are known to be relatively non-specific and require many factors also used in general DNA double-strand break repair, including both the Ku and catalytic subunits of the DNA-activated protein kinase (DNA-PK).

This talk will discuss recent developments in understanding both the cleavage and joining stages of the process. We have recently been able to reconstitute complete V(D)J recombination in a cell-free system. Incubation of a DNA substrate with the RAG proteins, followed by a second incubation with a HeLa cell fraction, leads to the formation of both coding joints and signal joints. The continued presence of the RAG proteins after cleavage is required to make coding joints, but inhibits the joining of RSS’s. The RAG proteins evidently influence the ability of other factors to act on coding ends and signal ends. Coding joints from the cell-free reaction often contain self-complementary “P nucleotide” tracts that arise from the asymmetric opening of hairpin ends, in analogy with similar insertions found in recombination in vivo.

In the cell-free reaction there is a strong preference for joining at sites of short DNA homologies, but addition of human DNA ligase I leads to a more diverse set of junctions, similar to those found in vivo. Homology-directed junctions have also been observed at high frequency in cells deficient in Ku protein, suggesting a direct link between Ku and the ligation step in double-strand break repair. We have obtained evidence for such a link with an oligonucleotide ligation assay. We find that Ku directly facilitates intermolecular ligation, particularly when ligation of blunt or near-blunt ends is required. Ku is able to bridge two DNA molecules, and the stimulation of ligation is probably due to this bridging. Thus Ku has an activity in DNA repair that is independent of its role as a cofactor for DNA-PK.

In cells, V(D)J recombination intermediates may also resolve by re-joining coding ends to signal ends (open-and-shut and hybrid joints). We show that these non-standard products can be generated in vitro by the RAG proteins alone, without the aid of ligase. This indicates that RAG-mediated cleavage of DNA is reversible, and is consistent with in vivo evidence that hybrid joints are not dependent on general double-strand break repair. Formation of hybrid joints is analogous to the dis-integration reaction of HIV integrase, strengthening the previously suggested relation between the biochemistry of the RAG proteins, retroviral integrases, and other transposons.