Mutagenesis, Vol. 15, No. 3, 281-286,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Retroviral expression of the NBS1 gene in cultured Nijmegen breakage syndrome cells restores normal radiation sensitivity and nuclear focus formation
Molecular Genetics Program, Virginia Mason Research Center, Seattle, WA 98101 and Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA and 1 Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands
The majority of cases of the autosomal recessive disorder Nijmegen breakage syndrome (NBS) are associated with null mutations in the NBS1 gene, which encodes a 95 kDa protein, nibrin. Cell lines established from NBS patients fail to express nibrin and display hypersensitivity to ionizing radiation and dysregulation of the nuclear localization of two key proteins involved in DNA repair, Mre11 and Rad50. Conclusive proof that mutations in the NBS1 gene are responsible for NBS requires that re-expression of normal nibrin in NBS cells complements these phenotypes. In the current study, retroviral expression vectors containing a normal copy of the NBS1 gene or a mutated form derived from a NBS patient were introduced into a well- characterized NBS cell line. Introduction of a normal copy of the NBS1 gene, but not the mutant form, resulted in robust expression of nibrin that displayed correct nuclear localization. Expression of nibrin also restored the ability of nibrin, Mre11 and Rad50 to complex and to redistribute within the nucleus in response to ionizing radiation. Radiation sensitivity of NBS cells expressing wild-type nibrin was restored to normal levels. Hence, introduction of the NBS1 gene can correct the phenotypes observed in NBS cells.
2 To whom correspondence should be addressed at: Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101, USA. Tel: +1 206 223 6476; Fax: +1 206 625 7213; Email: patcon{at}u.washington.edu
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