An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

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 t...

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Bibliographic Details
Published in:Molecular biology of the cell 2012-06, Vol.23 (11), p.2213-2225
Main Authors: Lin, I-Ting, Chao, Ju-Lan, Yao, Meng-Chao
Format: Article
Language:English
Subjects:
Antigens, Nuclear - metabolism
Base Sequence
Chromosome Breakage
Conjugation, Genetic
DNA Breaks
DNA Repair - genetics
DNA, Protozoan - genetics
DNA-Binding Proteins - metabolism
Gene Rearrangement - genetics
Green Fluorescent Proteins - metabolism
Histones - metabolism
Ku Autoantigen
Macronucleus - metabolism
Methylation
Models, Biological
Nuclear Proteins - metabolism
Phosphoproteins - metabolism
Protein Transport
Protozoan Proteins - metabolism
Recombinant Fusion Proteins - metabolism
Tetrahymena thermophila - cytology
Tetrahymena thermophila - genetics
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Summary: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.
ISSN:1059-1524
1939-4586
DOI:10.1091/mbc.E11-11-0952