An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria
Gregory Coffey,Colin R Campbell +1 more
TLDR
Testing for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression shows consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.Abstract:
Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.read more
Citations
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Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria
Nilesh Kumar Sharma,Aurelio Reyes,Paula D. Green,Matthieu J. Caron,Marcelo G. Bonini,Donna M. Gordon,Ian J. Holt,Janine H. Santos +7 more
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Human DNA repair genes.
Amiram Ronen,Barry W. Glickman +1 more
TL;DR: The current status of what is known about the DNA repair genes is tabulated, limited to genes whose function is directly related to DNA repair.
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Role of tyrosyl-DNA phosphodiesterase (TDP1) in mitochondria
TL;DR: It is demonstrated that a fraction of the TDP1 encoded by the nuclear T DP1 gene localizes to mitochondria, and it is shown that mitochondrial base excision repair depends on TDP2 activity, and evidence that TDP 1 is required for efficient repair of oxidative damage in mitochondrial DNA is provided.
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DNA damage related crosstalk between the nucleus and mitochondria.
Mohammad Saki,Aishwarya Prakash +1 more
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Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.
Satish Kumar Tadi,Robin Sebastian,Sumedha Dahal,Ravi K. Babu,Bibha Choudhary,Sathees C. Raghavan +5 more
TL;DR: Repair of double-strand breaks in mammalian mitochondria depends on microhomology-mediated end joining (MMEJ), and the protein machinery involved in miitochondrial MMEJ includes CtIP, FEN1, ligase III, MRE11, and PARP1.
References
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Journal ArticleDOI
The Human DNA Ligase III Gene Encodes Nuclear and Mitochondrial Proteins
TL;DR: Evidence is provided that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes, and identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.
Journal ArticleDOI
Molecular and biochemical characterization of xrs mutants defective in Ku80.
B. K. Singleton,Anne Priestley,H. Steingrimsdottir,David A. Gell,Tracy Blunt,Stephen P. Jackson,Alan R. Lehmann,P. A. Jeggo +7 more
TL;DR: The results with these revertants strongly support the model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation, and previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity.
Journal ArticleDOI
Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants.
A. Errami,Vaughn V. Smider,W. K. Rathmell,Dong Ming He,E. A. Hendrickson,Małgorzata Z. Zdzienicka,Gilbert Chu +6 more
TL;DR: Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.