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

被引:110
作者
Riballo, E
Doherty, AJ
Dai, Y
Stiff, T
Oettinger, MA
Jeggo, PA [1 ]
Kysela, B
机构
[1] Univ Sussex, MRC, Cell Mutat Unit, Brighton BN1 9RR, E Sussex, England
[2] Univ Cambridge, Wellcome Trust Ctr Mol Mechanisms, Inst Med Res, Cambridge CB2 2XY, England
[3] Univ Cambridge, Dept Haematol, Cambridge CB2 2XY, England
[4] Massachusetts Gen Hosp, Dept Mol Biol, Boston, MA 02114 USA
关键词
D O I
10.1074/jbc.M103866200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who over-responded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show that wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase with distinct optimal requirements for adenylate complex formation versus rejoining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-sensitive activity to the protein consistent with the predictions of the structural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation with little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but confer marked radiosensitivity.
引用
收藏
页码:31124 / 31132
页数:9
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