title: An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila.
abstract: Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 112, Taiwan, Republic of China.
authors.forename: I-Ting
authors.initials: IT
authors.lastname: Lin
authors.forename: Ju-Lan
authors.initials: JL
authors.lastname: Chao
authors.forename: Meng-Chao
authors.initials: MC
authors.lastname: Yao
mesh_terms: D034961:Antigens, Nuclear / Q000378:metabolism*; D001483:Base Sequence; D019457:Chromosome Breakage; D003227:Conjugation, Genetic; D053960:DNA Breaks; D004260:DNA Repair / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015321:Gene Rearrangement / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D000072200:Ku Autoantigen; D048648:Macronucleus / Q000378:metabolism; D008745:Methylation; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000166:cytology / Q000235:genetics*
chemical_list: D034961:Antigens, Nuclear; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; C101880:Pdd1 protein, Tetrahymena; D010750:Phosphoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000072200:Ku Autoantigen