Molecular and biochemical characterization of new X-ray-sensitive hamster cell mutants defective in Ku80.
Nucleic Acids Res
; 26(19): 4332-8, 1998 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-9742232
ABSTRACT
Ku, a heterodimer of approximately 70 and approximately 80 kDa subunits, is a nuclear protein that binds to double-stranded DNA ends and is a component of the DNA-dependent protein kinase (DNA-PK). Cell lines defective in Ku80 belong to group XRCC5 of ionizing radiation-sensitive mutants. Five new independent Chinese hamster cell mutants, XR-V10B, XR-V11B, XR-V12B, XR-V13B and XR-V16B, that belong to this group were isolated. To shed light on the nature of the defect in Ku80, the molecular and biochemical characteristics of these mutants were examined. All mutants, except XR-V12B, express Ku80 mRNA, but no Ku80 protein could clearly be detected by immunoblot analysis in any of them. DNA sequence analysis of the Ku80 cDNA from these mutants showed a deletion of 252 bp in XR-V10B; a 6 bp deletion that results in a new amino acid residue at position 107 and the loss of two amino acid residues at positions 108 and 109 in XR-V11B; a missense mutation resulting in a substitution of Cys for Tyr at position 114 in XR-V13B; and two missense mutations in XR-V16B, resulting in a substitution of Met for Val at position 331 and Arg for Gly at position 354. All these mutations cause a similar, 5-7-fold, increase in X-ray sensitivity in comparison to wild-type cells, and a complete lack of DNA-end binding and DNA-PK activities. This indicates that all these mutations lead to loss of the Ku80 function due to instability of the defective protein.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Tolerância a Radiação
/
Proteínas Nucleares
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Linhagem Celular
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DNA Helicases
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Antígenos Nucleares
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Proteínas de Ligação a DNA
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Mutação
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
1998
Tipo de documento:
Article
País de afiliação:
Holanda