Browsing by Subject "Chromosomes."

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Open Access
Analysis of the function of the nuclear matrix-associated protein C1D
(1999) Bilican, Bilada
DNA Double-strand breaks (DSBs) are generated as intermediate stnictures during V(D)J recombination, as a consequence of oxidative metabolism, or can be induced by exogenous factors such as gamma irradiation and radiomimetic drugs. Mutational studies identified the serine/threonine kinase, DNA-PK, as an essential component of DNA DSB repair machinery. The activation of the multi-component DNA-PK complex requires either free DNA ends or an association with the nuclear-matrix associated protein CID, which facilitates the activation of DNA-PK in a DNA end-independent fashion. The activation of DNA-PK through its interaction with CID, joins an increasing body of evidence which suggests a role for higher order nuclear organisation in the orchestration of complex cellular processes such as transcription, RNA splicing, nucleotide excision repair, replication and double-strand DNA break repair. In this study, the yeast two hybrid system was employed to screen a B-cell cDNA library to identify the interacting proteins with CID, and the interactions determined were further characterised. It was found that, CID interacts specifically with the recombinational hotspot binding protein Translin and Translin associated factor X, TRAX, both in vitro and in vivo, providing evidence that C 1D may play a critical role in DNA repair and recombination. Interestingly, an interaction between TRAX and DNAPKcs has also been identified under in vivo conditions. Tlie interaction of TRAX with DNA-PKcs and C ID indicates a connection between DNA double-strand break repair, recombination, and dynamic nuclear architecture.
Open Access
Identification of the role of the nuclear matrix protein C1D in DNA repair and recombination
(2002) Erdemir, Tuba
The nuclear matrix protein C1D is an activator of the DNA dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DNA-DSBs). C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon γ-irradiation, suggesting that C1D may play a role in repair of DNA-DSBs in vivo. In an attempt to identify the possible biological functions of C1D and the nuclear matrix, two approaches were employed. One of the strategies was to apply yeast-two hybrid system to screen for polypeptides that interact with C1D. This screening revealed a number of cDNA clones that encode mainly proteins involved in recombination, DNA repair and transcription. Among the identified proteins, TRAX (Translin Associated protein X) was chosen for further analysis. Although, the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins including Translin, into nucleus. After cloning the full-length TRAX ORF into bacterial and mammalian expression vectors, the specificity of the interaction between C1D and TRAX was confirmed both in vivo and in vitro conditions. Notably, it was shown that C1D and TRAX interact in mammalian cells only after γ- irradiation. In addition, it was demonstrated that the induced interaction of TRAX and C1D is not due to the alterations in their subcellular localizations but possibly through post-translational modifications in response to γ-irradiation. The second strategy was identification of the S. cerevisiae C1D homolog. Because of the high homology between yeast and mammalian C1D proteins, S. cerevisiae was used as a model organism to reveal the function of Yc1d. YC1D gene was knocked-out in yeast and the phenotypic and functional consequences of disruption of the YC1D gene was analyzed. It was found that yc1d mutant strain was slightly sensitive to γ-irradiation and was defective in DNA-DSB repair, thus, raising the possibility that yeast C1D and human C1D might be functional homologs.