Biochemical characterization of the small isoform of Drosophila melanogaster RECQ5 helicase

Recently the gene encoding a member of the RecQ helicase family, RecQ5, was cloned from the fruit fly, Drosophila melanogaster [J.J. Sekelsky, M.H. Br odsky, G.M. Rubin and R.S. Hawley (1999) Nucleic Acids Res., 27, 3762-3769] . The Drosophila RecQ5 transcript is alternatively spliced, like its human counterpart, to yield three protein isoforms. Two of these isoforms are alm ost identical and have a predicted molecular weight of 54 kDa. The third is oform is larger and contains, in addition to the helicase domain shared by all three isoforms, a long highly charged C-terminal region. A small isofor m of the Drosophila RecQ5 protein (RECQ5) has been expressed in Escherichia coli and purified. The purified protein is a single-stranded DNA-stimulate d ATPase (dATPase) and a 3'-->5' DNA helicase. Hydrolysis of the nucleotide cofactor is required for unwinding activity and dATP supported the unwindi ng reaction better than other NTPs. The turnover number for the single-stra nded DNA-stimulated dATPase activity was 1380 min(-1), similar to1.5-fold h igher than that observed for the ATPase activity (900 min(-1)). The purifie d protein catalyzed unwinding of partial duplex substrates up to at least 9 3 bp, however, unwinding of an 89 bp blunt duplex substrate was not detecte d.