Characterization of the dna ligase iv and xrcc4 complex in dna double-strand break repair

Abstract

DNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage. The nonhomologous end-joining (NHEJ) pathway is the principal mechanism for repairing DSBs in mammalian cells. It is also required for V(D)J recombination. There are at least four essential proteins in this pathway. These include Ku protein, DNA-PKcs, and the DNA Ligase IV /XRCC4 (DNL IV /XRCC4) complex. This dissertation reports the determination of the quaternary structure of the DNL IV /XRCC4 complex, the mapping of a major human autoimmune epitope in XRCC4, the identification of DNAPKcs phosphorylation sites in XRCC4, and an investigation of the biochemical significance of XRCC4 phosph9rylation. Biochemical characterization shows that DNA Ligase IV and XRCC4 form a stable mixed heterotetramer. this is the·active fonn of the enzyme and is essential for in vitro DNA end joining in the presence of additional factors deriyed from cell extracts. Data shown here also demonstrate-that the DNL _IV /XRCC4 complex is a human autoantigen. The major autoimmune epitope maps to amino acids 251-266. This epitope coincid~~ with ~ever~i sites where'XRCC4_ is.ppteniially· modified in response to radiation or inflammation, _includi,ng a DNA~PKcs phosphorylation site at serine 260. Results raise the possibility that radiation-induced post-translational modifications contribute to development of an autoimmune response in susceptible individuals. Previous work has shown that DNA-PKcs kinase activity is required for NHEJ, but the critical physiological .target of this enzyme is not yet known. Current work shows that DNA-PKcs phosphorylates serine 318 ofXRCC4, in addition to the serine 260 site · described above. The p~esence of serine 260 increases phosphorylation at serine 31.8, suggesting that phosphorylation can occur sequentially. Mutation of serine 260 reduced DNA end-joining activity and sensitivity to the PI3 kinase inhibitor (L Y294002). These <lat~ provide preliminary evidence that phosphorylation ofXRCC4 by DNA-PKcs contributes to regulation of DNA repair.