Abstract
We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg2+ for the helicase activity were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.
Highlights
The unwinding of parent DNA strands is a prerequisite to basic genetic processes including DNA replication, DNA repair, recombination, and transcription [1, 2]
TIP49a Is Structurally Similar to the Bacterial RuvB—We found earlier that rat TIP49a showed a significant homology with RuvB, which is a bacterial recombination factor and possesses ATPase/DNA helicase activities [8, 9]
The RuvB-homologous regions in the TIP49a protein described above included Walker A and B motifs, which are responsible for ATP binding and ATP hydrolysis, and are characteristic of RNA and DNA helicases (Fig. 1)
Summary
Plasmids for Expression of Rat TIP49a Protein—An NdeI site was created at translation initiation site of the TIP49a cDNA isolated from rat liver cDNA library in gt11 [6], and a fragment from NdeI to BamHI (in pBluescript vector downstream from the TIP49a cDNA) including an entire TIP49a coding region was inserted into pET-3a vector [13]. The TIP49a was incubated at 37 °C for 30 min in A buffer (20 mM Tris/HCl (pH 7.5), 70 mM KCl, 2.5 mM MgCl2, 1.5 mM dithiothreitol, 0.1 mM ATP, and 1.25 mCi of [␥-32P]ATP). For the DNA helicase assay, the reaction mixture (20 l) contained 20 mM Tris/HCl (pH 7.5), 2 mM dithiothreitol, 50 mg/ml BSA, 0.5 mM MgCl2, 80 mM KCl, 1 mM ATP, and 10 ng of 32P-labeled helicase substrate. Reaction mixture (20 l) in a buffer (20 mM Tris/HCl (pH 7.5), 70 mM KCl, 5 mM magnesium acetate, 1.5 mM dithiothreitol, 10% glycerol, and 5 Ci of [␣-32P]ATP) was irradiated by a UV cross-linker, LS1500 (Funakoshi), from a distance of 2 cm at 4 °C for 20 min. The sections were reacted with rabbit anti-TIP49a antisera (1:800) diluted with 1% bovine serum albumin in phosphate-buffered saline for 3 h. Negative control sections were incubated with normal rabbit serum at a 1:800 dilution
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