Table 1.
Details of the constructs and expression systems used, and ssDNA binding characteristics of the purified proteins.
Figure 1.
Alignment of TRAX and translin sequences.
The amino acid sequences of the TRAX and translin proteins of human and drosophila (UniProtKB/Swiss-Prot Q99598, Q8INE1, Q15631 and Q7JVK6, respectively) were aligned by ClustalW [33]. For clarity, only sequences of human TRAX (TRAX) and human translin (translin) are shown. The identical amino acid residues are shaded and similar residues are boxed. The five putative DNA binding regions (B1–5) predicted by the DP-Bind server and the nuclear localization signal (NLS) on the TRAX sequence are identified. Also marked are five basic regions (B1–5) on translin sequence and its nuclear export signal (NES). The figure was prepared with ESPript [34].
Figure 2.
Cartoon showing close proximity of B2 and B3 motifs on human translin-TRAX heterodimer.
Different views (A,B) of cartoon showing B2 and B3 motifs (spheres) of translin (cyan) and TRAX (blue) in translin-TRAX heterodimer. The figure was prepared using atomic coordinates of translin-TRAX complex structure (PDB code, 3PJA) and PyMol suite [35].
Figure 3.
Gel filtration analyses of human translin and translin-TRAX complexes.
A) A profile overlay of Superdex 200 gel-filtration chromatography of human translin and translin-TRAX complexes. The major peaks corresponding to molecular masses of 295 kDa for the complexes and 236 kDa for the translin were used for DNA-binding assays. B) The translin/TRAX protein complexes purified by gel-filtration chromatography were adjudged on SDS-PAGE. Molecular weight markers, lane1; human translin, lane 2; translinB2-TRAXB3 complex, lane 3; translin-TRAXB3 complex, lane 4; translinB2-TRAXB2 complex, lane 5 and wild-type translin-TRAX complex, lane 6. Nearly equimolar stochiometry of translin and TRAX proteins in the heteromeric complexes could be estimated from the band intensities. Truncation in TRAX protein was observed on storage. The truncation site at the C-terminus was confirmed by N-terminal sequencing and MALDI-TOF analyses. Integrated intensities of both the TRAX bands were summed to estimate translin/TRAX stoichiometry.
Figure 4.
Circular dichroism (CD) analysis of the proteins in the wavelength range of 200–260 nm.
The characteristic negative bands of α-helical proteins at 222 nm and 208 nm are observed for all the proteins and complexes. The CD spectrum of human translin essentially overlaps with those of translin-TRAX complexes. For clarity, CD spectra is shown for a few of the protein/complexes. The CD spectra presented are of human translin (translin), human mutant translinB3 (translinB3), human translin-TRAX complex (translin-TRAX), and complexes of mutants of human TRAXB3 and translin (translin-TRAXB3 and translinB2-TRAXB3).
Figure 5.
DNA-binding activity of the proteins/complexes.
A) [γ-32P]-labeled Bcl-CL1 24-mer ssDNA (100 nM) was incubated with human translin (50 nM of octameric translin) and translin-TRAX complexes (100 nM of octameric translin-TRAX complex). The mixtures were resolved on the 4.5% native-PAGE in TBE buffer. Prior to EMSA analysis all the purified samples were treated with DNase1 and RNaseA overnight at 20°C followed by purification using Ni-IDA chelating sepharose column. Lane 1, only DNA; lane 2, human translin; lane 3, translin-TRAX complex; lanes 4 and 5, complexes of human translin with the B2 and B3 mutants of human TRAX (translin-TRAXB2 and translin-TRAXB3), respectively; lane 6, Blank; lane 7, basic-2 region mutant of human translin (translinB2); lanes 8, 9 and 10, complexes of translinB2 with human TRAX, TRAXB2 and TRAXB3 (translinB2-TRAX, translinB2-TRAXB2 and translinB2-TRAXB3), respectively. Two bands with retarded mobility, compared to DNA alone, were observed in active protein complexes. Band 1 presumably corresponded to octameric complex while band 2 with very weak intensity belonged to higher oilgomers. B) Unlabled Bcl-CL1 24-mer ssDNA (10 µM) was incubated with human translin (1 µM and 5 µM, lanes 2 and lane 3 respectively), wild-type translin-TRAX complex (2 µM and 10 µM, lanes 4 and 5 respectively), translinB2 mutant (1 µM and 5 µM, lanes 6 and 7 respectively), translinB2-TRAX (2 µM and 10 µM, lanes 8 and 9 respectively), translinB2-TRAXB2 (2 µM and 10 µM, lanes 10 and 11 respectively) and translinB2-TRAXB3 (2 µM and 10 µM, lanes 12 and 13 respectively), and the reaction mixtures were resolved on 1.5% agarose gel were stained with ethidium bromide. At higher concentrations the ssDNA binding activity of translinB2-TRAXB3 complex was weakly detectable with ethidium bromide. This was not observed in autoradiograms with low concentrations of radiolabeled DNA and the protein complex.
Figure 6.
Autoradiograph showing protein-DNA crosslinking.
Purified translin, translin-TRAX complex and BSA were mixed with [γ-32P]-labeled Bcl-CL1 24-mer ssDNA. The mixtures were irradiated with UV-laser imparting total of 2400 mJ energy of 248 nm wavelength. Post-irradiation, the translin-TRAX complex was disrupted and TRAX was purified using affinity tag. The protein∶DNA complexes (translin-DNA, lane 2; TRAX-DNA, lane 3, BSA∶DNA, lane 4) and DNA alone (lane 1) were resolved on 12% SDS-PAGE and dried gel was autoradiographed on X-ray film. The translin-DNA and TRAX-DNA complexes differ by expected 6 kDa in molecular mass. The BSA, used as a negative control, did not show presence of protein-DNA covalent complex in the post-irradiated mixture. The intensities of free DNA band in lane 1 and 4 are much weaker, as only 5 µL of the irradiated BSA∶DNA mixture was loaded, compared to 20 µL of the translin∶DNA mixtures.