Biochemistry of nonhomologous DNA end joining (NHEJ) and its role in V(D)J recombination.

摘要

V(D)J recombination is a process that progenitor immune cells utilize to assemble variable domain exons of immunoglobulins and T cell receptors. While the cleavage of chromatin DNA is carried out by a lymphoid specific recombinase—the RAG complex, the joining of the broken chromatin is accomplished by the ubiquitous nonhomologous DNA end joining (NHEJ) pathway. Here, we present the biochemical studies of several key proteins to V(D)J recombination and NHEJ.;Ku is a protein that has been hypothesized to recognize the broken DNA ends once a double strand DNA break is generated. Our equilibrium and kinetic studies indicate that Ku demonstrates DNA length-dependent cooperativity. This raises the possibility that Ku may function in maintaining DNA ends at proximity by cooperative interactions across a DNA break. Modeling of the interaction of Ku and DNA with a mathematical computer program confirmed our conclusion and suggested that Ku prefers internal than terminal DNA positions.;It was reported that inositol hexakisphosphate (IP6) stimulates DNA end joining reactions in a cell free system. Our studies indicated that IP6, but not IP3, associates with Ku, raising the possibility that IP6 might enhance in vitro end joining via the interaction with Ku.;Recently, a new protein critical to V(D)J recombination and the NHEJ pathway was reported and named Artemis. The Artemis gene was cloned from human patients with radiosensitive severe combined immune deficiency (RS-SCID) by positional cloning. Our biochemical studies indicated that Artemis can form a functional complex with DNA-PKcs, another essential protein for V(D)J recombination and the NHEJ pathway, and resolves the hairpin intermediate for V(D)J recombination and processes overhanging DNA for the NHEJ pathway. These results may explain the phenotypes of Artemis deficient patients and DNA-PKcs deficient animals.;Altogether, these studies addressed some of the biochemical properties of Ku, Artemis, and DNA-PKcs. They are important not only in understanding the detailed enzymatic steps of V(D)J recombination and the NHEJ pathway, but also in designing inhibitors of these enzymes to sensitize cells to DNA damaging agents for cancer treatment and therapeutic regimens of human disorders related to aberrant V(D)J recombination events.