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 buff er extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted, The sequence containe d helicase motifs, and showed homology with RuvB DNA helicase of Thermus th ermophilus and an open reading frame for an unknown 50.5 k protein of Sacch aromyces cerevisiae, p50 was expressed as a GST-fusion protein and antiseru m against the protein was generated. p50 was localized to the nuclear matri x by cell fractionation and immunoblotting, p50 bound to ATP-Sepharose bead s. Ultracentrifugation and gel filtration analyses showed that p50 in rat l iver and Xenopus egg mitotic extracts exists as large complexes correspondi ng to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antib odies 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 put ative DNA helicase is present in a wide variety of species ranging from yea st to mammals.