Browsing by Subject "NHEJ"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Elucidating the role of redox effects and the KU80 C-Terminal region in the regulation of the human DNA repair protein KU
    (2010-07-20T16:00:25Z) McNeil, Sara M.; Turchi, John J.; Harrington, Maureen A.; Georgiadis, Millie M.
    DNA double strand breaks (DSB) are among the most lethal forms of DNA damage and can occur as a result of ionizing radiation (IR), radiomimetic agents, endogenous DNA-damaging agents, etc. If left unrepaired DSB’s can cause cell death, chromosome translocation and carcinogenesis. In humans, DSB are repaired predominantly by the non-homologous end joining (NHEJ) pathway. Ku, a heterodimer consisting of Ku70 and Ku80, functions in the recognition step of this pathway through binding DNA termini. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to create the full DNA-PK heterotrimer. Formation of DNA-PK results in autophosphorylation as well as phosphorylation of downstream proteins of the NHEJ pathway. Previous work shows that the extreme C-terminus of Ku80 stimulates the kinase activity of DNA-PKcs, and Ku DNA binding is regulated as a function of redox via stimulation of a conformational change when oxidized resulting in a decrease in DNA binding activity. To further understand these methods of regulation of Ku and DNA-PK, a pair of mutants has been constructed; one consisting of full length Ku70 and truncated Ku80 (Ku70/80ΔC) lacking 182 C-terminal amino acids. The removal of these amino acids was shown to have little to no effect on the proteins expression, stability or DNA binding, as determined by SDS-PAGE, western blot analysis and electrophoretic mobility shift assay (EMSA). When oxidized Ku70/80ΔC showed a decrease in DNA binding similar to that seen in wild type, however when re-reduced the mutant did not recover to the same extent as wild type. A second mutant was constructed, containing amino acids 590-732 of Ku80 (Ku80CTR), to further understand the mechanism by which Ku80 C-terminus interacts with the rest of the Ku heterodimer. Possible protein-protein interactions were evaluated by Ni-NTA affinity, gel filtration chromatography, fluorescence polarization and two forms of protein-protein cross-linking. Ni-NTA agarose affinity, and gel filtration chromatography failed to reveal an interaction in the presence or absence of DNA. However, photo-induced cross-linking of unmodified proteins (PICUP) as well as EDC cross-linking demonstrated an interaction which was not affected by DNA. The work presented here demonstrates that the interaction between Ku80CTR and Ku is rather weak, but it does exist and plays a relatively large role in the NHEJ pathway.
  • Thumbnail Image
    Item
    Functional Analysis of Two Novel DNA Repair Factors, Metnase and Pso4
    (2008-10-13T18:49:36Z) Beck, Brian Douglas; Lee, Suk-Hee
    Metnase is a novel bifunctional protein that contains a SET domain and a transposase domain. Metnase contains sequence-specific DNA binding activity and sequence non-specific DNA cleavage activity, as well as enhances genomic integration of exogenous DNA. Although Metnase can bind specifically to DNA sequences containing a core Terminal Inverted Repeat sequence, this does not explain how the protein could function at sites of DNA damage. Through immunoprecipitation and gel shift assays, I have identified the Pso4 protein as a binding partner of Metnase both in vitro and in vivo. Pso4 is essential for cell survival in yeast, and cells containing a mutation in Pso4 show increased sensitivity to DNA cross-linking agents. In addition, the protein has sequence-independent DNA binding activity, favoring double-stranded DNA over single-stranded DNA. I demonstrated that the two proteins form a 1:1 stochiometric complex, and once formed, Metnase can localize to DNA damage foci as shown by knockdown of Pso4 protein using in vivo immunofluorescence. In conclusion, this shows that Metnase plays an indispensable role in DNA end joining, possibly through its cleavage activity and association with DNA Ligase IV.
  • Thumbnail Image
    Item
    The influence of the Ku80 carboxy-terminus on activation of the DNA-dependent protein kinase and DNA repair is dependent on the structure of DNA cofactors
    (2013-11) Woods, Derek S.; Turchi, John J.; Harrington, Maureen A.; Malkova, Anna L.; Takagi, Yuichiro
    In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.