A Rat RuvB-like Protein, TIP49a, Is a Germ Cell-enriched Novel DNA Helicase

• Published in: The Journal of biological chemistry 1999-05, Vol.274 (22), p.15329-15335
• Main Authors: Makino, Yasutaka, Kanemaki, Masato, Kurokawa, Yumiko, Koji, Takehiko, Tamura, Taka-aki
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - pharmacology; Animals; ATPases Associated with Diverse Cellular Activities; Blotting, Western; Carrier Proteins - analysis; Carrier Proteins - genetics; DNA - metabolism; DNA Helicases - genetics; DNA Helicases - metabolism; DNA, Single-Stranded - metabolism; Escherichia coli - genetics; Germ Cells - enzymology; Immunohistochemistry; Liver - enzymology; Magnesium - pharmacology; Male; Molecular Sequence Data; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Rats; Recombinant Proteins - genetics; Sequence Alignment; Spermatozoa - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.22.15329

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 inEscherichia 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.