Cellular and biochemical impact of a mutation in DNA ligase IV conferring clinical radiosensitivity

DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombi nation, and during brain development. We previously reported a homozygous m utation (R278H) in DNA ligase IV in a developmentally normal leukemia patie nt who over-responded to radiotherapy. The impact of this hypomorphic mutat ion has been evaluated using cellular, biochemical, and structural approach es. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show t hat wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase wi th distinct optimal requirements for adenylate complex formation versus rej oining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-se nsitive activity to the protein consistent with the predictions of the stru ctural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation w ith little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but co nfer marked radiosensitivity.