spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Erdemir, T.
Right arrow Articles by Yavuzer, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Erdemir, T.
Right arrow Articles by Yavuzer, U.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

DNA damage-dependent interaction of the nuclear matrix protein C1D with translin-associated factor X (TRAX)

Tuba Erdemir1,*, Bilada Bilican2,*, Dilhan Oncel1, Colin R. Goding2 and Ugur Yavuzer1,{ddagger}

1 Bilkent University, Molecular Biology and Genetics Department, 06533, Bilkent, Ankara, Turkey
2 Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK
* These authors contributed equally.



View larger version (11K):

[in a new window]
  		Fig. 1. C1D and TRAX interact specifically. TRAX fused to the Gal4 activation domain (AD-TRAX) was transformed into the reporter yeast strain together with DBD alone, DBD-lamin (La), DBD-Daughterless (Da) or DBD-C1D and interactions were measured by ß-galactosidase activity using ONPG.

 


View larger version (26K):

[in a new window]
  		Fig. 2. C1D and TRAX interact in vitro. C1D and hepatitis C virus core proteins (HCV-core/pQE) were expressed in bacteria using the plasmid pQE30 (Qiagen) and purified by Ni-NTA column under denaturing conditions. One µg of each of the immobilized fusion proteins and pQE alone were incubated with In vitro Transcribed and Translated (IVTT) TRAX product and pull-down assay was performed. One tenth of the input TRAX was bound by C1D.

 


View larger version (14K):

[in a new window]
  		Fig. 3. The putative LZ region of TRAX is essential for interactions with C1D and Translin. (A) Yeast two-hybrid assay was performed using DBD-C1D and an activation domain (AD) tagged forms of TRAX; wild-type (WT), N-terminal region containing the LZ motif (N-Ter) or mutLZ where the LZ region of TRAX has been mutated within an otherwise intact protein. The interactions were measured by ß-galactosidase activity using ONPG. (B) Constructs as in (A) were used to detect interaction with the Translin protein expressed as a fusion to the bacterial LexA protein (DBD-Translin). Mutations that disrupt the LZ region of TRAX abolish its interaction with Translin and neither the N-terminal region of TRAX carrying an intact LZ nor the C-terminal region alone (C-Ter) are sufficient for interaction with Translin.

 


View larger version (31K):

[in a new window]
  		Fig. 4. Binding of C1D and Translin to TRAX is mutually exclusive. C1D was expressed in bacteria and immobilized on Ni-NTA column as explained in the legend to Fig. 2. IVTT TRAX and Translin products were incubated with the immobilized C1D either alone (lanes 3 and 4) or together. TRAX (T) and Translin (t) IVTT products were incubated simultaneously (S) with the immobilized C1D for 2 hours before washing (lane 5), TRAX and Translin IVTT products were pre-incubated (PI) for 1 hour before adding to immobilized C1D and incubated for another hour (lane 6), IVTT product of TRAX was incubated with the immobilized C1D (C) for 1 hour and then Translin was added and incubated for an additional hour (lane 7). The plasmid expressing His only (pQE) was expressed in bacteria, purified and immobilized under the same conditions as C1D and was used in lane 8 as a negative control.

 


View larger version (45K):

[in a new window]
  		Fig. 5. SDS-resistant C1D homodimers interact with TRAX upon {gamma}-irradiation. (A) Transiently transfected COS 7 cells were immunoprecipitated using the antibodies indicated. For lanes 3-6 the cells were treated with 20 Gy of ionizing irradiation before lysate preparation. Western blotting was performed by an anti-C1D antibody. The monomer and dimer forms of C1D are indicated. (B) Transiently transfected COS 7 cells were immunoprecipitated using the indicated antibodies. For lane 3-7, the cells were treated with 50 j/m2 UV irradiation. The top panel shows the western blotting using an anti-C1D antibody, and in the bottom panel, the blot was stripped and reprobed with an anti-GFP antibody to demonstrate the presence of GFP-TRAX. For each lane, immunoprecipitations were performed using the cell lysates obtained from 3x105 cells.

 


View larger version (31K):

[in a new window]
  		Fig. 6. Subcellular localizations of TRAX, Translin and C1D. (A) Plasmids expressing CFP-Translin, YFP-TRAX and RFP-C1D were transfected into COS 7 cells and subcellular localizations were determined in the living cells. Translin was mainly cytoplasmic; however, 6% of cells exhibited nuclear/cytoplasmic staining (a). TRAX was nuclear (b) and C1D was mainly nuclear, but a cytoplasmic staining was also evident (c). (B) COS 7 cells were double transfected with the indicated plasmids. (a-c) TRAX/C1D double-transfected cells showing that TRAX is solely nuclear (a) whereas C1D is showing a nuclear/cytoplasmic staining pattern (b), and colocalizes with TRAX in the nucleus (c). In TRAX/Translin double-transfected cells (d-f), TRAX and Translin colocalize in the cytoplasm and in the nucleus. The middle cell in d and f expresses TRAX only. In C1D/Translin double-transfected cells (g-i), C1D is mainly nuclear (g) and Translin is cytoplasmic (h). (C) COS 7 cells triple transfected with TRAX-, Translin- and C1D-expressing plasmids. (a-c) A single cell in which a diffuse nuclear/cytoplasmic staining pattern is observed. In d-f, another field is shown where two of the cells were triple transfected and in which the upper cell TRAX and C1D were expressed in the nucleus (d and f) when Translin was mainly cytoplasmic (e); the lower cell shows a diffuse nuclear-cytoplasmic staining pattern as in a-c. (D) Summary of the results demonstrating the percentage of nuclear/cytoplasmic and solely nuclear localizations of Translin, TRAX and C1D.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002