Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase.
Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.
Department of Biochemistry, Faculty of Science, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101, Japan.
N
N
Kikuchi
T
T
Gohshi
S
S
Kawahire
T
T
Tachibana
Y
Y
Yoneda
T
T
Isobe
C R
CR
Lim
K
K
Kohno
T
T
Ichimura
S
S
Omata
T
T
Horigome
D000074183:ATPases Associated with Diverse Cellular Activities; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000235:genetics
D001426:Bacterial Proteins; C061260:RuvB protein, Bacteria; D000255:Adenosine Triphosphate; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486550:Ruvbl1 protein, rat