Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA.
Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.
He
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Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Lee
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Hendrickson
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EA
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004265:DNA Helicases; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D015329:Gene Rearrangement, T-Lymphocyte; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D011836:Radiation Tolerance / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D059005:Topoisomerase II Inhibitors
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen