Ku80-deficient Cells Exhibit Excess Degradation of Extrachromosomal DNA*

Mammalian cells possess a protein complex, termed DNA-PK, which binds to DNA double strand breaks in vitro. The complex consists of the heterodimeric Ku autoantigen and a DNA-dependent protein kinase, DNA-PKcs. Cell lines that are deficient for components of this complex are sensitive to ionizing radiation and have impaired V(D)J recombination, a site-specific recombination process. We have tested these cell lines for their ability to repair double strand breaks in transfected DNA. The xrs-6 cell line, which is deficient for the 80-kDa subunit of the Ku autoantigen, exhibited reduced stability of transfected DNA. Prior to obvious reductions in DNA stability, the levels of homologous recombination and DNA end joining were unaffected. However, the recovery of end joining products with precisely joined ends was reduced, with a concomitant increase in products containing deletions. Unlike the Ku80-deficient cells, no reduction in DNA stability was detected in DNA-PKcs-deficient scid cells. Scid cells also exhibited normal levels of homologous recombination and DNA end joining. These experiments implicate the Ku autoantigen, but not DNA-PKcs, in a direct role in protecting DNA ends from degradation.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  M. Jasin,et al.  Genetic manipulation of genomes with rare-cutting endonucleases. , 1996, Trends in genetics : TIG.

[3]  Christina A. Cuomo,et al.  Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps , 1995, Cell.

[4]  M. Jasin,et al.  Studies on the Influence of Cytosine Methylation on DNA Recombination and End-joining in Mammalian Cells (*) , 1995, The Journal of Biological Chemistry.

[5]  B. Dujon,et al.  Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[6]  F. Alt,et al.  Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation , 1995, Cell.

[7]  M. Oettinger,et al.  DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect , 1995, Science.

[8]  P. Jeggo,et al.  DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Bollag,et al.  Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.

[11]  J. Dick,et al.  Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents. , 1994, Science.

[12]  M. Lieber,et al.  Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. , 1994, Science.

[13]  F. Alt,et al.  Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. , 1994, Science.

[14]  J. Phillips,et al.  Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome , 1994, Molecular and cellular biology.

[15]  G. Chu,et al.  A DNA end-binding factor involved in double-strand break repair and V(D)J recombination , 1994, Molecular and cellular biology.

[16]  P. Rouet,et al.  Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Anderson,et al.  DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. , 1994, The Journal of biological chemistry.

[18]  C. Anderson,et al.  DNA damage and the DNA-activated protein kinase. , 1993, Trends in biochemical sciences.

[19]  F. Alt,et al.  Impairment of V(D)J recombination in double-strand break repair mutants. , 1993, Science.

[20]  J. Brown,et al.  Characterization of the DNA double strand break repair defect in scid mice. , 1993, Cancer research.

[21]  S. Jackson,et al.  The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen , 1993, Cell.

[22]  M. Lieber,et al.  Analysis of the defect in DNA end joining in the murine scid mutation , 1992, Molecular and cellular biology.

[23]  P. Jeggo,et al.  Use of damaged plasmid to study DNA repair in X-ray sensitive (xrs) strains of Chinese hamster ovary (CHO) cells. , 1989, International journal of radiation biology.

[24]  J. Thacker,et al.  Gene recombination in X-ray-sensitive hamster cells , 1987, Molecular and cellular biology.

[25]  J A Hardin,et al.  Mechanism of interaction between Ku protein and DNA. , 1986, The Journal of biological chemistry.

[26]  R. Kucherlapati,et al.  Homologous recombination in a Chinese hamster X-ray-sensitive mutant. , 1986, Mutation research.

[27]  J. Steitz,et al.  Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. , 1986, The Journal of biological chemistry.

[28]  E. Dziubałtowska,et al.  Genotoxic effects of ethyl acrylate and methyl acrylate in the mouse evaluated by the micronucleus test. , 1984, Mutation research.

[29]  A. S. Waldman,et al.  Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI. , 1994, Nucleic acids research.

[30]  A. Carroll,et al.  The SCID mouse mutant: definition, characterization, and potential uses. , 1991, Annual review of immunology.