A rat RuvB-like protein, TIP49a, is a germ cell-enriched novel DNA helicase.
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.
Makino
Y
Y
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
Kanemaki
M
M
Kurokawa
Y
Y
Koji
T
T
Tamura
T a
Ta
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005854:Germ Cells / Q000201:enzymology*; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D013094:Spermatozoa / Q000201:enzymology
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486550:Ruvbl1 protein, rat; D008274:Magnesium